Antibodies recognizing tau

ABSTRACT

The invention provides antibodies that specifically bind tau. The antibodies inhibit or delay tau-associated pathologies and associated symptomatic deterioration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC 119(e) of U.S.Provisional Application No. 62/330,800 filed May 2, 2016, which isincorporated by reference in its entirety for all purposes

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in file 497448SEQLIST.txt is 59 kilobytes,was created on May 2, 2017, and is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Tau is a well-known human protein that can exist in phosphorylated forms(see, e.g., Goedert, Proc. Natl. Acad. Sci. U.S.A. 85:4051-4055(1988);Goedert, EMBO J. 8:393-399(1989); Lee, Neuron 2:1615-1624(1989);Goedert, Neuron 3:519-526(1989); Andreadis, Biochemistry31:10626-10633(1992). Tau has been reported to have a role instabilizing microtubules, particularly in the central nervous system.Total tau (t-tau, i.e., phosphorylated and unphosphorylated forms) andphospho-tau (p-tau, i.e., phosphorylated tau) are released by the brainin response to neuronal injury and neurodegeneration and have beenreported to occur at increased levels in the CSF of Alzheimer's patientsrelative to the general population (Jack et al., Lancet Neurol 9: 119-28(2010)).

Tau is the principal constituent of neurofibrillary tangles, whichtogether with plaques are a hallmark characteristic of Alzheimer'sdisease. The tangles constitute abnormal fibrils measuring 10 nm indiameter occurring in pairs wound in a helical fashion with a regularperiodicity of 80 nm. The tau within neurofibrillary tangles isabnormally phosphorylated (hyperphosphorylated) with phosphate groupsattached to specific sites on the molecule. Severe involvement ofneurofibrillary tangles is seen in the layer II neurons of theentorhinal cortex, the CA1 and subicular regions of the hippocampus, theamygdala, and the deeper layers (layers III, V, and superficial VI) ofthe neocortex in Alzheimer's disease. Hyperphosphorylated tau has alsobeen reported to interfere with microtubule assembly, which may promoteneuronal network breakdown.

Tau inclusions are part of the defining neurophathology of severalneurodegenerative diseases including Alzheimer's disease, frontotemporallobar degeneration, progressive supranuclear palsy and Pick's disease.

BRIEF SUMMARY OF THE CLAIMED INVENTION

In one aspect, the invention provides an isolated monoclonal antibodythat specifically binds to tau. Examples of such antibodies bind to anepitope within amino acid residues 55-78, 60-75 or 61-70 of SEQ ID NO:3(corresponding to amino acid residues 113-136, 118-133, or 119-128,respectively, of SEQ ID NO:1).

Some such antibodies compete for binding to human tau with antibody16G7. Some such antibodies bind to the same epitope on human tau as16G7.

Some antibodies comprise three light chain CDRs as and three heavy chainCDRs of monoclonal antibody 16G7, wherein 16G7 is a mouse antibodycharacterized by a heavy chain variable region having an amino acidsequence comprising SEQ ID NO: 7 and a light chain variable regionhaving an amino acid sequence comprising SEQ ID NO: 11. In someantibodies, the three heavy chain CDRs are as defined by Kabat/ChothiaComposite (SEQ ID NOs: 8, 9, and 10) and the three light chain CDRs areas defined by Kabat/Chothia Composite (SEQ ID NOs: 12, 13, and 14).

For example, the antibody can be 16G7 or a chimeric, veneered, orhumanized form thereof. In some such antibodies, the variable heavychain has 85% identity to human sequence. In some such antibodies, thevariable light chain has 85% identity to human sequence. In some suchantibodies, each of the variable heavy chain and variable light chainhas 85% identity to human germline sequence. In some such antibodies,the mature heavy chain variable region comprises the three heavy chainCDRs are as defined by Kabat/Chothia Composite (SEQ ID NOs: 8, 9, and10) and the three light chain CDRs are as defined by Kabat/ChothiaComposite (SEQ ID NOs: 12, 13, and 14); provided that position H31 isoccupied by S or G, position H60 is occupied by N or A and position 64is occupied by K or Q. In some such antibodies, CDR-H1 has an amino acidsequence comprising SEQ ID NO: 49. In some such antibodies, CDR-H2 hasan amino acid sequence comprising SEQ ID NO: 50. In some suchantibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO: 51.In some such antibodies, the antibody is a humanized antibody.

In some such antibodies, the CDR-H1 has an amino acid sequencecomprising SEQ ID NO: 49 and CDR-H2 has an amino acid sequencecomprising SEQ ID NO: 50. In some such antibodies, the CDR-H1 has anamino acid sequence comprising SEQ ID NO: 49 and CDR-H2 has an aminoacid sequence comprising SEQ ID NO: 51.

Some antibodies are a humanized or chimeric 16G7 antibody thatspecifically binds to human tau, wherein 16G7 is a mouse antibodycharacterized by a mature heavy chain variable region of SEQ ID NO:7 anda mature light chain variable region of SEQ ID NO: 11. Some suchantibodies are a humanized antibody comprising a humanized mature heavychain variable region comprising the three heavy chain CDRs of 16G7 anda humanized mature light chain variable region comprising the threelight chain CDRs of 16G7. In some such antibodies, the CDRs are of adefinition selected from the group of Kabat, Chothia, Kabat/ChothiaComposite, AbM and Contact.

In some such antibodies the humanized mature heavy chain variable regioncomprises the three Kabat/Chothia Composite heavy chain CDRs of 16G7(SEQ ID NOs: 8-10) and the humanized mature light chain variable regioncomprises the three Kabat/Chothia Composite light chain CDRs of 16G7(SEQ ID NOs: 12-14).

In some such antibodies, the humanized mature heavy chain variableregion comprises the three Kabat heavy chain CDRs of 16G7 (SEQ ID NO:39,SEQ ID NO:9, and SEQ ID NO:10) and the humanized mature light chainvariable region comprises the three Kabat light chain CDRs of 16G7 (SEQID NOs: 12-14).

In some such antibodies, the humanized mature heavy chain variableregion comprises the three Chothia heavy chain CDRs of 16G7 (SEQ IDNO:40, SEQ ID NO:41, and SEQ ID NO:10) and the humanized mature lightchain variable region comprises the three Chothia light chain CDRs of16G7 (SEQ ID NOs: 12-14).

In some such antibodies, the humanized mature heavy chain variableregion comprises the three AbM heavy chain CDRs of 16G7 (SEQ ID NO:8,SEQ ID NO:42, and SEQ ID NO:10)) and the humanized mature light chainvariable region comprises the three AbM light chain CDRs of 16G7 (SEQ IDNOs: 12-14).

In some such antibodies, the humanized mature heavy chain variableregion comprises the three Contact heavy chain CDRs of 16G7 (SEQ IDNO:46-48) and the humanized mature light chain variable region comprisesthe three Contact light chain CDRs of 16G7 (SEQ ID NO:43-45).

In some antibodies, the humanized mature heavy chain variable region hasan amino acid sequence at least 90% identical to any one of SEQ IDNO:15-27 and a humanized mature light chain variable region having anamino acid sequence at least 90% identical to any one of SEQ ID NO:28-30.

In some such antibodies, at least one of the following positions in theVH region is occupied by the amino acid as specified: H1 is occupied byE, H5 is occupied by Q, H7 is occupied by P, H9 is occupied by S, H10 isoccupied by V, H11 is occupied L, H12 is occupied by V, H13 is occupiedby R, H20 is occupied by L, H40 is occupied by R, H48 is occupied by I,H66 is occupied by K, H67 is occupied by A, H69 is occupied by L, H71 isoccupied by V, H73 is occupied by I, H75 is occupied by S, H80 isoccupied by V, H82a is occupied by T, H82b is occupied by S, H83 isoccupied by T, and H85 is occupied by E. In some such antibodies,positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H40, H48, H66, H67,H69, H71, H73, H75, H80, H82a, H82b, H83, and H85 in the VH region areoccupied by, E, Q, P, S, V, L, V, R, L, R, I, K, A, L, V, I, S, V, T, S,T, and E, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H1 is occupied by E,H5 is occupied by Q, H7 is occupied by P, H9 is occupied by S, H10 isoccupied by V, H11 is occupied by L, H12 is occupied by V, H13 isoccupied by R, H20 is occupied by L, H48 is occupied by I, H69 isoccupied by L, H71 is occupied by V, H82a is occupied by T, H82b isoccupied by S, H83 is occupied by T, and H85 is occupied by E. In someantibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H48, H69,H71, H82a, H82b, H83, and H85 in the VH region are occupied by E, Q, P,S, V, L, V, R, L, I, L, V, T, S, T, and E, respectively.

In some antibodies at least one of the following positions in the VHregion is occupied by the amino acid as specified: H1 is occupied by E,H5 is occupied by Q, H11 is occupied by L, H12 is occupied by V, H20 isoccupied by L, H48 is occupied by I, H69 is occupied by L, H71 isoccupied by V, H82a is occupied by T, H82b is occupied by S, H83 isoccupied by T, and H85 is occupied by E. In some antibodies, H1, H5,H11, H12, H20, H48, H69, H71, H82a, H82b, H83, and H85 in the VH regionare occupied by E, Q, L, V, L, I, L, V, T, S, T, and E, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H1 is occupied by E,H12 is occupied by V, H31 is occupied by G, H40 is occupied by R, H48 isoccupied by I, H60 is occupied by A, H69 is occupied by L, H73 isoccupied by I, and H83 is occupied by T. In some antibodies, positionsH1, H12, H31, H40, H48, H60, H69, H73, and H83 in the VH region areoccupied by are occupied by E, V, G, R, I, A, L, I, and T, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H1 is occupied by E,H12 is occupied by V, H40 is occupied by R, H48 is occupied by I, H69 isoccupied by L, H73 is occupied by I, and H83 is occupied by T. In someantibodies, positions H1, H12, H40, H48, H69, H73, and H83 in the VHregion are occupied by are occupied by E, V, R, I, L, I, and T,respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H1 is occupied by E,H12 is occupied by V, H40 is occupied by R, H48 is occupied by I, H66 isoccupied K, H67 is occupied by A, H69 is occupied by L, H71 is occupiedby V, H73 is occupied by I, and H83 is occupied by T. In someantibodies, positions H1, H12, H40, H48, H66, H67, H69, H71, H73, andH83 in the VH region are occupied by are occupied by E, V, R, I, K, A,L, V, I, and T, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H1 is occupied by E,H12 is occupied by V, H40 is occupied by R, H48 is occupied by I, H69 isoccupied by L, H73 is occupied by I, and H83 is occupied by T. In someantibodies, positions H1, H12, H40, H48, H69, H73, and H83 in the VHregion are occupied by are occupied by E, V, R, I, L, I, and T,respectively. In some antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H7 is occupied by P, H71 is occupied by V, and H73 is occupied by I. Insome antibodies, positions H7, H71, and H73 in the VH region areoccupied by are occupied by P, V, and I, respectively. In someantibodies, at least one of the following positions in the VH region isoccupied by the amino acid as specified: H31 is occupied by G and H60 isoccupied by A. In some antibodies, positions H31 and H60 in the VHregion are occupied by G and A, respectively. In some antibodies, atleast one of the following positions in the VH region is occupied by theamino acid as specified: H1 is occupied by Q or E, H5 is occupied by Vor Q, H7 is occupied by S or P, H9 is occupied by A or S, H10 isoccupied by E or V, H11 is occupied V or L, H12 is occupied by K or V,H13 is occupied by K or R, H20 is occupied by V or L, H31 is occupied byG or S, H37 is occupied by V or A, H 38 is occupied by R or K, H40 isoccupied by A or R, H48 is occupied by M or I, H60 is occupied by A orN, H64 is occupied by Q or K, H66 is occupied by R or K, H67 is occupiedby V or A, H69 is occupied by M or L, H71 is occupied by R or V, H73 isoccupied by T or I, H75 is occupied by I, S, or A, H80 is occupied by Mor V, H82a is occupied by S or T, H82b is occupied by R or S, H83 isoccupied by R or T, and H85 is occupied by D or E. In some antibodies,positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H37, H38, H40, H48,H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83, and H85 in the VHregion are occupied by E, Q, P, S, V, L, V, R, L, A, K, R, I, K, A, L,V, I, S, V, T, S, T, and E, respectively. In some antibodies, positionsH1, H5, H11, H12, H20, H48, H69, H71, H75, H82a, H82b, H83, and H85 inthe VH region are occupied by E, Q, L, V, L, I, L, V, A, T, S, T, and E,respectively. In some antibodies, positions H1, H12, H40, H48, H66, H67,H69, H71, H73, and H83 in the VH region are occupied by E, V, R, I, K,A, L, V, I, and T, respectively. In some antibodies, positions H1, H12,H31, H40, H48, H60, H66, H67, H69, H71, H73, and H83 in the VH regionare occupied by E, V, G, R, I, A, K, A, L, V, I, and T, respectively. Insome antibodies, positions H1, H12, H40, H48, H69, H73, and H83 in theVH region are occupied by E, V, R, I, L, I, and T, respectively. In someantibodies, positions H1, H12, H31, H40, H48, H60, H66, H67, H69, H71,H73, and H83 in the VH region are occupied by Q, V, G, R, I, A, K, A, L,V, I, and T, respectively. In some antibodies, positions H1, H7 and H73in the VH region are occupied by Q, P, V and I, respectively. In someantibodies, positions H1, H12, H31, H40, H48, H60, H64, H69, H73, andH83 in the VH region are occupied by, E, V, G, R, I, A, Q, L, I, and T,respectively. In some antibodies, positions H1, H5, H7, H9, H10, H11,H12, H13, H20, H48, H69, H71, H75, H82a, H82b, H83, and H85 in the VHregion are occupied by E, Q, P, S, V, L, V, R, L, I, L, V, A, T, S, T,and E. In some antibodies, positions H1, H5, H11, H12, H20, H48, H66,H67, H69, H71, H73, H75, H82a, H82b, H83, and H85 in the VH region areoccupied by, E, Q, L, V, L, I, K, A, L, V, I, S, T, S, T, and E. In someantibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H38, H40,H48, H66, H67, H69, H71, H73I, H75, H80, H82a, H82b, H83, and H85 in theVH region are occupied by E, Q, P, S, V, L, V, R, L, K, R, I, K, A, L,V, I, S, V, T, S, T, and E, respectively. In some antibodies, positionsH1, H5, H7, H9, H10, H11, H12, H13, H20, H40, H48, H66, H67, H69, H71,H73, H75, H80, H82a, H82b, H83, and H85 in the VH region are occupied byQ, P, S, V, L, V, R, L, R, I, K, A, L, V, I, S, V, T, S, T, and E,respectively. In some antibodies, positions H7, H71, and H73 in the VHregion are occupied by P, V, and I, respectively. In some antibodies,positions H71 in the VH region is occupied by V. In some antibodies,positions H1, H7, H71, and H73 in the VH region are occupied by E, P, V,and I.

In some antibodies, at least one of the following positions in the VLregion is occupied by the amino acid as specified: L4 is occupied by L,L9 is occupied by S, L15 is occupied by V, L22 is occupied by S, and L43is occupied by S. In some antibodies, positions L4, L9, L15, L22, andL43 in the VL region are occupied by L, S, V, S, and S, respectively.

In some antibodies, at least one of the following positions in the VLregion is occupied by the amino acid as specified: L4 is occupied by L,L9 is occupied by S, L15 is occupied by V, L18 is occupied by K, L19 isoccupied by V, L21 is occupied by M, L22 is occupied by S, and L43 isoccupied by S. In some antibodies, L4, L9, L15, L18, L19, L21, L22, andL43 in the VL region are occupied by L, S, V, K, V, M, S, and S,respectively.

In some antibodies, at least one of the following positions in the VLregion is occupied by the amino acid as specified: L4 is occupied by Mor L, L9 is occupied by D or S, L15 is occupied by L or V, L18 isoccupied by R or K, L19 is occupied by A or V, L21 is occupied by I orM, L22 is occupied by N or S, L43 is occupied by P or S, and L48 isoccupied by I or M. In some antibodies, L4, L9, L15, L18, L19, L21, L22,and L43 in the VL region are occupied by L, S, V, K, V, M, S, and S,respectively.

In some antibodies, positions L4, L9, L15, L22, and L43 in the VL regionare occupied by L, S, V, S, and S, respectively. In some antibodies,positions L4, L9, L15, L18, L19, L21, L22, and L43 in the VL region areoccupied by L, S, V, K, V, M, S, and S, respectively.

Some antibodies comprise a mature heavy chain variable region having anamino acid sequence at least 95% identical to any one of SEQ ID NO:15-27 and a mature light chain variable region having an amino acidsequence at least 95% identical to any one of SEQ ID NO: 28-30. Someantibodies comprise a mature heavy chain variable region having an aminoacid sequence at least 98% identical to any one of SEQ ID NO: 15-27 anda mature light chain variable region having an amino acid sequence atleast 98% identical to any one of SEQ ID NO: 28-30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of any of SEQ ID NO:15-27 and the mature light chainvariable region has an amino acid sequence of any one of SEQ IDNO:28-30. In some antibodies, the mature heavy chain variable region hasan amino acid sequence of SEQ ID NO:15 and the mature light chainvariable region has an amino acid sequence of SEQ ID NO:28. In someantibodies, the mature heavy chain variable region has an amino acidsequence of SEQ ID NO:15 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO:29. In some antibodies, the matureheavy chain variable region has an amino acid sequence of SEQ ID NO:15and the mature light chain variable region has an amino acid sequence ofSEQ ID NO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:16 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:16 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:16 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:17 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:17 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:17 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:18 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region region has an amino acid sequence ofSEQ ID NO:18 and the mature light chain variable region has an aminoacid sequence of SEQ ID NO:29. In some antibodies, the mature heavychain variable region has an amino acid sequence of SEQ ID NO:18 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:19 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:19 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:19 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:20 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:20 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:20 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:21 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:21 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:21 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region an amino acidsequence of SEQ ID NO:22 and the mature light chain variable region hasan amino acid sequence of SEQ ID NO:28. In some antibodies, the matureheavy chain variable region has an amino acid sequence of SEQ ID NO:22and the mature light chain variable region has an amino acid sequence ofSEQ ID NO:29. In some antibodies, the mature heavy chain variable regionhas an amino acid sequence of SEQ ID NO:22 and the mature light chainvariable region has an amino acid sequence of SEQ ID NO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:23 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:23 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:23 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:24 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:24 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:24 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:25 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:25 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:25 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:26 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:26 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:26 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:27 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:28. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:27 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:27 and themature light chain variable region has an amino acid sequence of SEQ IDNO:30.

For example, the antibody can be a chimeric antibody. For example, theantibody can be a veneered antibody.

The antibody can be an intact mouse, chimeric, veneered or humanizedantibody or a binding fragment, single-chain antibody Fab fragment,Fab′2 fragment, or single chain Fv. Some of the antibodies have a humanIgG1 isotype, while others may have a human IgG2 or IgG4 isotype. Someantibodies have the mature light chain variable region fused to a lightchain constant region and the mature heavy chain variable region fusedto a heavy chain constant region. The heavy chain constant region ofsome antibodies is a mutant form of a natural human heavy chain constantregion which has reduced binding to a Fcγ receptor relative to thenatural human heavy chain constant region.

Some antibodies may have at least one mutation in the constant region,such as a mutation that reduces complement fixation or activation by theconstant region, for example, a mutation at one or more of positions241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331 by EUnumbering. Some antibodies have an alanine at positions 318, 320 and322. Some antibodies can be at least 95% w/w pure. The antibody can beconjugated to a therapeutic, cytotoxic, cytostatic, neurotrophic, orneuroprotective agent.

In another aspect, the invention provides a pharmaceutical compositioncomprising any of the antibodies disclosed herein and apharmaceutically-acceptable carrier.

In another aspect, the invention provides a nucleic acid encoding theheavy chain and/or light chain of any of the antibodies disclosedherein, a recombinant expression vector comprising the nucleic acid anda host cell transformed with the recombinant expression vector.

In yet another aspect, the invention provides methods of humanizing anynon-human antibody described herein, for example, mouse antibodies 16G7,wherein 16G7 is characterized by a mature heavy chain variable region ofSEQ ID NO:7 and a mature light chain variable region of SEQ ID NO:11.Such methods can involve selecting one or more acceptor antibodies,synthesizing a nucleic acid encoding a humanized heavy chain comprisingCDRs of the mouse heavy chain and a nucleic acid encoding a humanizedlight chain comprising CDRs of the mouse antibody light chain, andexpressing the nucleic acids in a host cell to produce a humanizedantibody.

Methods of producing antibodies, such as a humanized, chimeric orveneered antibody, for example humanized, chimeric or veneered forms of16G7, are also provided. In such methods, cells transformed with nucleicacids encoding the heavy and light chains of the antibody are culturedso that the cells secrete the antibody. The antibody can then bepurified from the cell culture media.

Cell lines producing any of the antibodies disclosed herein can beproduced by introducing a vector encoding heavy and light chains of theantibody and a selectable marker into cells, propagating the cells underconditions to select for cells having increased copy number of thevector, isolating single cells from the selected cells; and bankingcells cloned from a single cell selected based on yield of antibody.

Some cells can be propagated under selective conditions and screened forcell lines naturally expressing and secreting at least 100 mg/L/10⁶cells/24 hours. Single cells can be isolated from the selected cells.Cells cloned from a single cell can then be banked Single cells can beselected based on desirable properties, such as the yield of theantibody. Exemplary cell lines are cell lines expressing 16G7.

The invention also provides methods of inhibiting or reducingaggregation of tau in a subject having or at risk of developing atau-mediated amyloidosis, comprising administering to the subject aneffective regime of an antibody disclosed herein, thereby inhibiting orreducing aggregation of tau in the subject. Exemplary antibodies includehumanized versions of 16G7.

Also provided are methods of treating or effecting prophylaxis of atau-related disease in a subject, comprising administering an effectiveregime of an antibody disclosed herein and thereby treating or effectingprophylaxis of the disease. Examples of such a disease are Alzheimer'sdisease, Down's syndrome, mild cognitive impairment, primary age-relatedtauopathy, postencephalitic parkinsonism, posttraumatic dementia ordementia pugilistica, Pick's disease, type C Niemann-Pick disease,supranuclear palsy, frontotemporal dementia, frontotemporal lobardegeneration, argyrophilic grain disease, globular glial tauopathy,amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam,corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy bodyvariant of Alzheimer disease (LBVAD), or progressive supranuclear palsy(PSP). In some methods, the tau-related disease is Alzheimer's disease.In some methods the patient is an ApoE4 carrier.

Also provided are methods of reducing aberrant transmission of taucomprising administering an effective regime of an antibody disclosedherein and thereby reducing transmission of tau.

Also provided are methods of inducing phagocytosis of tau comprisingadministering an effective regime of an antibody disclosed herein andthereby inducing phagocytosis of tau.

Also provided are methods of inhibiting tau aggregation or depositioncomprising administering an effective regime of an antibody disclosedherein thereby inhibiting tau aggregation or deposition.

Also provided are methods of inhibiting formation of tau tanglescomprising administering an effective regime of an antibody disclosedherein.

The invention also provides a method of detecting tau protein depositsin a subject having or at risk of a disease associated with tauaggregation or deposition comprising administering to a subject anantibody disclosed herein, and detecting the antibody bound to tau inthe subject. Examples of such a disease are Alzheimer's disease, Down'ssyndrome, mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP). Insome embodiments, the antibody is administered by intravenous injectioninto the body of the subject. In some embodiments, the antibody isadministered directly to the brain of the subject by intracranialinjection or by drilling a hole through the skull of the subject. Insome embodiments, the antibody is labeled. In some embodiments, theantibody is labeled with a fluorescent label, a paramagnetic label, or aradioactive label. In some embodiments, the radioactive label isdetected using positron emission tomography (PET) or single-photonemission computed tomography (SPECT).

The invention also provides a method of measuring efficacy of treatmentin a subject being treated for a disease associated with tau aggregationor deposition, comprising measuring a first level of tau proteindeposits in the subject prior to treatment by administering to a subjectan antibody disclosed herein, and detecting a first amount of theantibody bound to tau in the subject, administering the treatment to thesubject, measuring a second level of tau protein deposits in the subjectafter treatment by administering to a subject the antibody, anddetecting the antibody bound to tau in the subject, wherein a decreasein the level of tau protein deposits indicates a positive response totreatment.

The invention also provides a method of measuring efficacy of treatmentin a subject being treated for a disease associated with tau aggregationor deposition, comprising measuring a first level of tau proteindeposits in the subject prior to treatment by administering to a subjectan antibody disclosed herein, and detecting a first amount of antibodybound to tau in the subject, administering the treatment to the subject,measuring a second level of tau protein deposits in the subject aftertreatment by administering to a subject the antibody, and detecting asecond amount of antibody bound to tau in the subject, wherein no changein the level of tau protein deposits or a small increase in tau proteindeposits indicates a positive response to treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of experiments designed to map the epitope(s)bound by the murine 16G7 monoclonal antibody.

FIG. 2 depicts an alignment of heavy chain variable regions of the mouse16G7 antibody and humanized versions of the 16G7 antibody. The CDRs asdefined by Kabat/Chothia Composite are in boldface. Positions in heavychain variable regions of the mouse 16G7 antibody and humanized versionsof the 16G7 antibody where amino acid residues differ from the“Majority” sequence are boxed.

FIG. 3 depicts an alignment of light chain variable regions of the mouse16G7 antibody and humanized versions of the 16G7 antibody. The CDRs asdefined by Kabat are in boldface. Positions in light chain variableregions of the mouse 16G7 antibody and humanized versions of the 16G7antibody where amino acid residues differ from the “Majority” sequenceare boxed.

FIGS. 4A and 4B depict results of experiments showing that 16G7immunocaptures tau from human Alzheimer's disease tissue.

FIG. 5 depicts results of disaggregation assays for selected mousemonoclonal anti-tau antibodies.

FIG. 6 depicts affinity of mouse 16G7 antibody to tau.

FIG. 7 depicts binding kinetics for chimeric 16G7 antibody and humanized16G7 antibodies VHV2a, VHV2b, and VHV6a H7L2 towards tau.

FIG. 8 depicts avidity of mouse 16G7 Fab fragment and mouse 16G7 intactantibody towards aggregated tau.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-8).

SEQ ID NO:2 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-7).

SEQ ID NO:3 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-6), (4RON human tau).

SEQ ID NO:4 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-5).

SEQ ID NO:5 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-4).

SEQ ID NO:6 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-2).

SEQ ID NO: 7 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 16G7 antibody.

SEQ ID NO: 8 sets forth the amino acid sequence of Kabat/ChothiaComposite CDR-H1 of the mouse 16G7 antibody.

SEQ ID NO:9 sets forth the amino acid sequence of Kabat CDR-H2 of themouse 16G7 antibody.

SEQ ID NO: 10 sets forth the amino acid sequence of Kabat CDR-H3 of themouse 16G7 antibody.

SEQ ID NO: 11 sets forth the amino acid sequence of the light chainvariable region of the mouse 16G7 antibody.

SEQ ID NO: 12 sets forth the amino acid sequence of Kabat CDR-L1 of themouse 16G7 antibody.

SEQ ID NO: 13 sets forth the amino acid sequence of Kabat CDR-L2 of themouse 16G7 antibody.

SEQ ID NO: 14 sets forth the amino acid sequence of Kabat CDR-L3 of themouse 16G7 antibody.

SEQ ID NO:15 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv1.

SEQ ID NO:16 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv2.

SEQ ID NO:17 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv2a.

SEQ ID NO:18 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv2aB7.

SEQ ID NO:19 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv2b.

SEQ ID NO:20 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv2bH7.

SEQ ID NO:21 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv3.

SEQ ID NO:22 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv4.

SEQ ID NO:23 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv5V.

SEQ ID NO:24 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv5VR.

SEQ ID NO:25 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv6a.

SEQ ID NO:26 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv6b.

SEQ ID NO:27 sets forth the amino acid sequence of heavy chain variableregion of the humanized 16G7 antibody hu16G7VHv7.

SEQ ID NO:28 sets forth the amino acid sequence of the light chainvariable region of the humanized 16G7 antibody hu16G7VLv1.

SEQ ID NO:29 sets forth the amino acid sequence of the light chainvariable region of the humanized 16G7 antibody hu16G7VLv2.

SEQ ID NO:30 sets forth the amino acid sequence of the light chainvariable region of the humanized 16G7 antibody hu16G7VLv3.

SEQ ID NO:31 sets forth the amino acid sequence of the heavy chainvariable acceptor Acc.# AAA18265.1.

SEQ ID NO:32 sets forth the amino acid sequence of the heavy chainvariable acceptor Acc.# ADW08092.1.

SEQ ID NO:33 sets forth the amino acid sequence of the heavy chainvariable acceptor Acc.# IMGT# IGHV1-2*02.

SEQ ID NO:34 sets forth the amino acid sequence of the light chainvariable acceptor Acc.# AAB24404.1.

SEQ ID NO:35 sets forth the amino acid sequence of the light chainvariable acceptor Acc.# AEK69364.1.

SEQ ID NO:36 sets forth the amino acid sequence of the light chainvariable acceptor Acc.# IMGT#IGKV4-1*01.

SEQ ID NO: 37 sets forth a nucleic acid sequence encoding the heavychain variable region of the mouse 16G7 antibody.

SEQ ID NO: 38 sets forth a nucleic acid sequence encoding the lightchain variable region of the mouse 16G7 antibody.

SEQ ID NO: 39 sets forth the amino acid sequence of Kabat CDR-H1 of themouse 16G7 antibody.

SEQ ID NO: 40 sets forth the amino acid sequence of Chothia CDR-H1 ofthe mouse 16G7 antibody.

SEQ ID NO: 41 sets forth the amino acid sequence of Chothia CDR-H2 ofthe mouse 16G7 antibody.

SEQ ID NO: 42 sets forth the amino acid sequence of AbM CDR-H2 of themouse 16G7 antibody.

SEQ ID NO: 43 sets forth the amino acid sequence of Contact CDR-L1 ofthe mouse 16G7 antibody.

SEQ ID NO: 44 sets forth the amino acid sequence of Contact CDR-L2 ofthe mouse 16G7 antibody.

SEQ ID NO: 45 sets forth the amino acid sequence of Contact CDR-L3 ofthe mouse 16G7 antibody.

SEQ ID NO: 46 sets forth the amino acid sequence of Contact CDR-H1 ofthe mouse 16G7 antibody.

SEQ ID NO: 47 sets forth the amino acid sequence of Contact CDR-H2 ofthe mouse 16G7 antibody.

SEQ ID NO: 48 sets forth the amino acid sequence of Contact CDR-H3 ofthe mouse 16G7 antibody.

SEQ ID NO: 49 sets forth the amino acid sequence of an alternateKabat-Chothia Composite CDR-H1 of a humanized 16G7 antibody.

SEQ ID NO: 50 sets forth the amino acid sequence of an alternate KabatCDR-H2 of a humanized 16G7 antibody.

SEQ ID NO: 51 sets forth the amino acid sequence of an alternate KabatCDR-H2 of a humanized 16G7 antibody.

SEQ ID NO:52 sets forth the consensus amino acid sequence among theheavy chain variable regions of the mouse 16G7 and humanized 16G7antibodies (labeled “Majority’ in FIG. 2).

SEQ ID NO:53 sets forth the consensus amino acid sequence between thelight chain variable regions of the mouse 16G7 and humanized 16G7antibodies (labeled “Majority’ in FIG. 3).

SEQ ID NO: 54 sets forth the amino acid sequence of the heavy chain of achimeric 16G7 antibody.

SEQ ID NO: 55 sets forth the amino acid sequence of the light chain of achimeric 16G7 antibody.

Definitions

Monoclonal antibodies or other biological entities are typicallyprovided in isolated form. This means that an antibody or otherbiologically entity is typically at least 50% w/w pure of interferingproteins and other contaminants arising from its production orpurification but does not exclude the possibility that the monoclonalantibody is combined with an excess of pharmaceutically acceptablecarrier(s) or other vehicle intended to facilitate its use. Sometimesmonoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/wpure of interfering proteins and contaminants from production orpurification. Often an isolated monoclonal antibody or other biologicalentity is the predominant macromolecular species remaining after itspurification.

Specific binding of an antibody to its target antigen means an affinityand/or avidity of at least 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² M⁻¹.Specific binding is detectably higher in magnitude and distinguishablefrom non-specific binding occurring to at least one unrelated target.Specific binding can be the result of formation of bonds betweenparticular functional groups or particular spatial fit (e.g., lock andkey type) whereas nonspecific binding is usually the result of van derWaals forces. Specific binding does not however necessarily imply thatan antibody binds one and only one target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. See generally,Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7 (incorporated by reference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region (also referred toherein as a “light chain variable domain” (“VL domain”) or “heavy chainvariable domain” (“VH domain”), respectively) consists of a “framework”region interrupted by three “complementarity determining regions” or“CDRs.” The framework regions serve to align the CDRs for specificbinding to an epitope of an antigen. The CDRs include the amino acidresidues of an antibody that are primarily responsible for antigenbinding. From amino-terminus to carboxyl-terminus, both VL and VHdomains comprise the following framework (FR) and CDR regions: FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain arealso referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3;CDRs 1, 2, and 3 of a VH domain are also referred to herein,respectively, as CDR-H1, CDR-H2, and CDR-H3.

The assignment of amino acids to each VL and VH domain is in accordancewith any conventional definition of CDRs. Conventional definitionsinclude, the Kabat definition (Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), the Chothia definition (Chothia & Lesk, J. Mol. Biol.196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); acomposite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothiaand Kabat CDRs; the AbM definition used by Oxford Molecular's antibodymodelling software; and, the contact definition of Martin et al(bioinfo.org.uk/abs) (see Table 1). Kabat provides a widely usednumbering convention (Kabat numbering) in which corresponding residuesbetween different heavy chains or between different light chains areassigned the same number. When an antibody is said to comprise CDRs by acertain definition of CDRs (e.g., Kabat) that definition specifies theminimum number of CDR residues present in the antibody (i.e., the KabatCDRs). It does not exclude that other residues falling within anotherconventional CDR definition but outside the specified definition arealso present. For example, an antibody comprising CDRs defined by Kabatincludes among other possibilities, an antibody in which the CDRscontain Kabat CDR residues and no other CDR residues, and an antibody inwhich CDR H1 is a composite Chothia-Kabat CDR H1 and other CDRs containKabat CDR residues and no additional CDR residues based on otherdefinitions.

TABLE 1 Conventional Definitions of CDRs Using Kabat Numbering Compositeof Loop Kabat Chothia Chothia & Kabat AbM Contact L1 L24 - - - L34L24 - - - L34 L24 - - - L34 L24 - - - L34 L30 - - - L36 L2 L50 - - - L56L50 - - - L56 L50 - - - L56 L50 - - - L56 L46 - - - L55 L3 L89 - - - L97L89 - - - L97 L89 - - - L97 L89 - - - L97 L89 - - - L96 H1 H31 - - -H35B H26 - - - H32 . . . H34* H26 - - - H35B* H26 - - - H35B H30 - - -H35B H2 H50 - - - H65 H52 - - - H56 H50 - - - H65 H50 - - - H58H47 - - - H58 H3 H95 - - - H102 H95 - - - H102 H95 - - - H102 H95 - - -H102 H93 - - - H101 *CDR-H1 by Chothia can end at H32, H33, or H34(depending on the length of the loop). This is because the Kabatnumbering scheme places insertions of extra residues at 35A and 35B,whereas Chothia numbering places them at 31A and 31B. If neither H35Anor H35B (Kabat numbering) is present, the Chothia CDR-H1 loop ends atH32. If only H35A is present, it ends at H33. If both H35A and H35B arepresent, it ends at H34.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target includingseparate heavy chains, light chains Fab, Fab′, F(ab′)₂, F(ab)c, Dabs,nanobodies, and Fv. Fragments can be produced by recombinant DNAtechniques, or by enzymatic or chemical separation of intactimmunoglobulins. The term “antibody” also includes a bispecific antibodyand/or a humanized antibody. A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites (see, e.g., Songsivilai and Lachmann,Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol.,148:1547-53 (1992)). In some bispecific antibodies, the two differentheavy/light chain pairs include a humanized 16G7 heavy chain/light chainpair and a heavy chain/light chain pair specific for a different epitopeon tau than that bound by 16G7.

In some bispecific antibodies, one heavy chain/light chain pair is ahumanized 16G7 antibody as further disclosed below and the other heavychain/light chain pair is from an antibody that binds to a receptorexpressed on the blood brain barrier, such as an insulin receptor, aninsulin-like growth factor (IGF) receptor, a leptin receptor, or alipoprotein receptor, or a transferrin receptor (Friden et al., Proc.Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science259:373-377, 1993). Such a bispecific antibody can be transferred crossthe blood brain barrier by receptor-mediated transcytosis. Brain uptakeof the bispecific antibody can be further enhanced by engineering thebi-specific antibody to reduce its affinity to the blood brain barrierreceptor. Reduced affinity for the receptor resulted in a broaderdistributioin in the brain (see, e.g., Atwal et al., Sci. Trans. Med. 3,84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).

Exemplary bispecific antibodies can also be: (1) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg(2010)); (2) a Tandab, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (3) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(4) a so-called “dock and lock” molecule, based on the “dimerization anddocking domain” in Protein Kinase A, which, when applied to Fabs, canyield a trivalent bispecific binding protein consisting of two identicalFab fragments linked to a different Fab fragment; or (5) a so-calledScorpion molecule, comprising, e.g., two scFvs fused to both termini ofa human Fc-region. Examples of platforms useful for preparing bispecificantibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2(F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab armexchange, Genmab).

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids (also known aslinear epitopes) are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding (also known asconformational epitopes) are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined X-ray crystallography of theantibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% as measured in acompetitive binding assay. Some test antibodies inhibit binding of thereferences antibody by at least 75%, 90% or 99%. Antibodies identifiedby competition assay (competing antibodies) include antibodies bindingto the same epitope as the reference antibody and antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

The term “pharmaceutically acceptable” means that the carrier, diluent,excipient, or auxiliary is compatible with the other ingredients of theformulation and not substantially deleterious to the recipient thereof.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,and situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “biological sample” refers to a sample of biological materialwithin or obtainable from a biological source, for example a human ormammalian subject. Such samples can be organs, organelles, tissues,sections of tissues, bodily fluids, peripheral blood, blood plasma,blood serum, cells, molecules such as proteins and peptides, and anyparts or combinations derived therefrom. The term biological sample canalso encompass any material derived by processing the sample. Derivedmaterial can include cells or their progeny. Processing of thebiological sample may involve one or more of filtration, distillation,extraction, concentration, fixation, inactivation of interferingcomponents, and the like.

The term “control sample” refers to a biological sample not known orsuspected to include tau-related disease-affected regions, or at leastnot known or suspect to include diseased regions of a given type.Control samples can be obtained from individuals not afflicted with thetau-related disease. Alternatively, control samples can be obtained frompatients afflicted with the tau-related disease. Such samples can beobtained at the same time as a biological sample thought to comprise thetau-related disease or on a different occasion. A biological sample anda control sample can both be obtained from the same tissue. Preferably,control samples consist essentially or entirely of normal, healthyregions and can be used in comparison to a biological sample thought tocomprise tau-related disease-affected regions. Preferably, the tissue inthe control sample is the same type as the tissue in the biologicalsample. Preferably, the tau-related disease-affected cells thought to bein the biological sample arise from the same cell type (e.g., neurons orglia) as the type of cells in the control sample.

The term “disease” refers to any abnormal condition that impairsphysiological function. The term is used broadly to encompass anydisorder, illness, abnormality, pathology, sickness, condition, orsyndrome in which physiological function is impaired, irrespective ofthe nature of the etiology.

The term “symptom” refers to a subjective evidence of a disease, such asaltered gait, as perceived by the subject. A “sign” refers to objectiveevidence of a disease as observed by a physician.

The term “positive response to treatment” refers to a more favorableresponse in an individual patient or average response in a population ofpatients relative to an average response in a control population notreceiving treatment.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesinsubstantial variations, such as values within a standard margin oferror of measurement (e.g., SEM) of a stated value.

Statistical significance means p≤0.05.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

DETAILED DESCRIPTION I. General

The invention provides antibodies that bind to tau. Some antibodiesspecifically bind to an epitope within residues 55-78 of SEQ ID NO:3(corresponding to residues 113-136 of SEQ ID NO:1), such as, forexample, an epitope within residues 60-75 (corresponding to residues118-133 of SEQ ID NO:1) or within residues 61-70 (corresponding toresidues 119-128 of SEQ ID NO:1). Some such antibodies can specificallybind to two peptides, for example, a first peptide comprising residues55-69 of SEQ ID NO:3 (corresponding to residues 113-127 of SEQ ID NO:1)as well as a second peptide comprising residues 64-78 (corresponding toresidues 122-136 of SEQ ID NO:1). Some antibodies bind to tauirrespective of phosphorylation state. Some antibodies of the inventionserve to inhibit or delay tau-associated pathologies and associatedsymptomatic deterioration. Although an understanding of mechanism is notrequired for practice of the invention, a reduction in toxicity mayoccur as a result of the antibody inducing phagocytosis of tau,inhibiting tau from inter or intramolecular aggregation, or from bindingto other molecules, by stabilizing a non-toxic conformation, byinhibiting intercellular or intracellular transmission of pathogenic tauforms, by blockade of tau phosphorylation, by preventing binding of tauto cells, or by inducing proteolytic cleavage of tau, among othermechanisms. The antibodies of the invention or agents that induce suchantibodies can be used in methods of treating or effecting prophylaxisof Alzheimer's and other diseases associated with tau.

II. Target Molecules

Unless otherwise apparent from the context, reference to tau means anatural human form of tau including all isoforms irrespective of whetherposttranslational modification (e.g., phosphorylation, glycation, oracetylation) is present. There are six major isoforms (splice variants)of tau occurring in the human brain. The longest of these variants has441 amino acids, of which the initial met residue is cleaved. Residuesare numbered according to the 441 isoform. Thus, for example, referenceto a phosphorylation at position 404 means position 404 of the 441isoform, or corresponding position of any other isoform when maximallyaligned with the 441 isoform. The amino acid sequences of the isoformsand Swiss-Prot numbers are indicated below.

P10636-8 (SEQ ID NO: 1)        10         20         30         40         50         60 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG        70         80         90        100        110        120 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG       130        140        150        160        170        180 HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK       190        200        210        220        230        240 TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK       250        260        270        280        290        300 SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV       310        320        330        340        350        360 PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI       370        380        390        400        410        420 THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV       430        440  DSPQLATLAD EVSASLAKQG L P10636-7 (SEQ ID NO: 2)        10         20         30         40         50         60 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG        70         80         90        100        110        120 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT       130        140        150        160        170        180 KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR       190        200        210        220        230        240 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ       250        260        270        280        290        300 PGGGKVQIIN KKLDLSNVQS KCGSKDNIKH VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH       310        320        330        340        350        360 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG       370        380        390        400        410 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GLP10636-6 (4R0N human tau) (SEQ ID NO: 3)        10         20         30         40         50         60 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA        70         80         90        100        110        120 AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA       130        140        150        160        170        180 PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS       190        200        210        220        230        240 AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQII NKKLDLSNVQ SKCGSKDNIK       250        260        270        280        290        300 HVPGGGSVQI VYKPVDLSKV TSKCGSLGNI HHKPGGGQVE VKSEKLDFKD RVQSKIGSLD       310        320        330        340        350        360 NITHVPGGGN KKIETHKLTF RENAKAKTDH GAEIVYKSPV VSGDTSPRHL SNVSSTGSID       370        380  MVDSPQLATL ADEVSASLAK QGL P10636-5 (SEQ ID NO: 4)        10         20         30         40         50         60 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG        70         80         90        100        110        120 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG       130        140        150        160        170        180 HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK       190        200        210        220        230        240TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK       250        260        270        280        290        300SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIVYK PVDLSKVTSK CGSLGNIHHK       310        320        330        340        350        360PGGGQVEVKS EKLDFKDRVQ SKIGSLDNIT HVPGGGNKKI ETHKLTFREN AKAKTDHGAE       370        380        390        400        410IVYKSPVVSG DTSPRHLSNV SSTGSIDMVD SPQLATLADE VSASLAKQGL P10636-4(SEQ ID NO: 5)        10         20         30         40         50         60MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG        70         80         90        100        110        120SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT       130        140        150        160        170        180KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR       190        200        210        220        230        240SRTPSLPTPP TREPKKVAVV RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ       250        260        270        280        290        300PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI       310        320        330        340        350        360THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV       370        380  DSPQLATLAD EVSASLAKQG L P10636-2 (SEQ ID NO: 6)10         20         30         40         50         60MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA        70         80         90        100        110        120AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGOANA TRIPAKTPPA       130        140        150        160        170        180PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS       190        200        210        220        230        240AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH       250        260        270        280        290        300HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG       310        320        330        340        350AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL

Reference to tau includes known natural variations about 30 of which arelisted in the Swiss-Prot database and permutations thereof, as well asmutations associated with tau pathologies, such as dementia, Pick'sdisease, supranuclear palsy, etc. (see, e.g., Swiss-Prot database andPoorkaj, et al. Ann Neurol. 43:815-825 (1998)). Some examples of taumutations numbered by the 441 isoform are a lysine to threonine mutationat amino acid residue 257 (K257T), an isoleucine to valine mutation atamino acid position 260 (1260V); a glycine to valine mutation at aminoacid position 272 (G272V); an asparagine to lysine mutation at aminoacid position 279 (N279K); an asparagine to histidine mutation at aminoacid position 296 (N296H); a proline to serine mutation at amino acidposition 301 (P301S); a proline to leucine mutation at amino acid 301(P301L); a glycine to valine mutation at amino acid position 303(G303V); a serine to asparagine mutation at position 305 (S305N); aglycine to serine mutation at amino acid position 335 (G335S); a valineto methionine mutation at position 337 (V337M); a glutamic acid tovaline mutation at position 342 (E342V); a lysine to isoleucine mutationat amino acid position 369 (K3691); a glycine to arginine mutation atamino acid position 389 (G389R); and an arginine to tryptophan mutationat amino acid position 406 (R406W).

Tau can be phosphorylated at one or more amino acid residues includingtyrosine at amino acid positions 18, 29, 97, 310, and 394 serine atamino acid positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238,262, 293, 324, 356, 396, 400, 404, 409, 412, 413, and 422; and threonineat amino acids positions 175, 181, 205, 212, 217, 231, and 403. Unlessotherwise apparent from context, reference to tau, or their fragmentsincludes the natural human amino acid sequences including isoforms,mutants, and allelic variants thereof.

IV. Antibodies

A. Binding Specificity and Functional Properties

The invention provides antibodies that bind to tau. Some antibodiesspecifically bind to an epitope within residues 55-78 of SEQ ID NO:3(corresponding to residues of 113-136 of SEQ ID NO:1), such as, forexample, an epitope within residues 60-75 of SEQ ID NO:3 (correspondingto residues 118-133 of SEQ ID NO:1) or within residues 61-70 of SEQ IDNO:3 (corresponding to residues 119-128 of SEQ ID NO:1). Some antibodiesbind to tau irrespective of phosphorylation state. These antibodies canbe obtained by immunizing with a tau polypeptide purified from a naturalsource or recombinantly expressed. Antibodies can be screened forbinding tau in unphosphorylated form as well as a form in which one ormore residues susceptible to phosphorylation are phosphorylated. Suchantibodies preferably bind with indistinguishable affinities or at leastwithin a factor of 1.5, 2 or 3-fold to phosphorylated tau compared tonon-phosphorylated tau (i.e., are “pan-specific”). 16G7 is an example ofa pan-specific monoclonal antibody. The invention also providesantibodies binding to the same epitope as any of the foregoingantibodies, such as, for example, the epitope of 16G7. Also included areantibodies competing for binding to tau with any of the foregoingantibodies, such as, for example, competing with 16G7.

The above-mentioned antibodies can be generated de novo by immunizingwith a peptide including residues 55-78, 55-69, or 64-78 of SEQ ID NO: 3(corresponding to residues 113-136, 113-127, or 122-136, respectively,of SEQ ID NO:1) or by immunizing with a full length tau polypeptide orfragment thereof comprising such residues and screening for specificbinding to a peptide including such residues. Such peptides arepreferably attached to a heterologous conjugate molecule that helpselicit an antibody response to the peptide. Attachment can be direct orvia a spacer peptide or amino acid. Cysteine is used as a spacer aminoacid because its free SH group facilitates attachment of a carriermolecule. A polyglycine linker (e.g., 2-6 glycines), with or without acysteine residue between the glycines and the peptide can also be used.The carrier molecule serves to provide a T-cell epitope that helpselicit an antibody response against the peptide. Several carriers arecommonly used particularly keyhole limpet hemocyanin (KLH), ovalbuminand bovine serum albumin (BSA). Peptide spacers can be added to peptideimmunogen as part of solid phase peptide synthesis. Carriers aretypically added by chemical cross-linking Some examples of chemicalcrosslinkers that can be used include cross-N-maleimido-6-aminocaproylester or m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see forexample, Harlow, E. et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. 1988; Sinigaglia etal., Nature, 336:778-780 (1988); Chicz et al., J. Exp. Med., 178:27-47(1993); Hammer et al., Cell 74:197-203 (1993); Falk K. et al.,Immunogenetics, 39:230-242 (1994); WO 98/23635; and, Southwood et al. J.Immunology, 160:3363-3373 (1998)). The carrier and spacer if present canbe attached to either end of the immunogen.

A peptide with optional spacer and carrier can be used to immunizelaboratory animals or B-cells as described in more detail below.Hybridoma supernatants can be tested for ability to bind one or morepeptides including residues 55-78, 55-69, or 64-78 of SEQ ID NO: 3(corresponding to residues 113-136, 113-127, or 122-136, respectively,of SEQ ID NO:1) and/or phosphorylated and non-phosphorylated forms oftau, such as, for example, a full-length isoform of tau with position404 in phosphorylated form. The peptide can be attached to a carrier orother tag to facilitate the screening assay. In this case, the carrieror tag is preferentially different than the combination of spacer andcarrier molecule used for immunization to eliminate antibodies specificfor the spacer or carrier rather than the tau peptide. Any of the tauisoforms can be used.

The invention provides monoclonal antibodies binding to epitopes withintau. An antibody designated 16G7 is one such exemplary mouse antibody.Unless otherwise apparent from context, reference to 16G7 should beunderstood as referring to any of the mouse, chimeric, veneered, andhumanized forms of this antibody. The antibody has been deposited as[DEPOSIT NUMBER]. This antibody specifically binds within amino acidresidues 55-78, 55-69, or 64-78 of SEQ ID NO: 3 (corresponding toresidues 113-136, 113-127, or 122-136, respectively, of SEQ ID NO:1).This antibody is further characterized by its ability to bind bothphosphorylated and unphosphorylated tau, both non-pathological andpathological forms and conformations of tau, and misfolded/aggregatedforms of tau.

Some antibodies of the invention bind to the same or overlapping epitopeas an antibody designated 16G7. The sequences of the heavy and lightchain mature variable regions of this antibody are designated SEQ ID NO:7 and SEQ ID NO:11, respectively. Other antibodies having such a bindingspecificity can be produced by immunizing mice with tau or a portionthereof including the desired epitope and screening resulting antibodiesfor binding to tau optionally in competition with an antibody having thevariable regions of mouse 16G7 (IgG1 kappa). Fragments of tau includingthe desired epitope can be linked to a carrier that helps elicit anantibody response to the fragment and/or be combined with an adjuvantthe helps elicit such a response. Such antibodies can be screened fordifferential binding to tau or a fragment thereof compared with mutantsof specified residues. Screening against such mutants more preciselydefines the binding specificity to allow identification of antibodieswhose binding is inhibited by mutagenesis of particular residues andwhich are likely to share the functional properties of other exemplifiedantibodies. The mutations can be systematic replacement substitutionwith alanine (or serine if an alanine is present already) one residue ata time, or more broadly spaced intervals, throughout the target orthroughout a section thereof in which an epitope is known to reside. Ifthe same set of mutations significantly reduces the binding of twoantibodies, the two antibodies bind the same epitope.

Antibodies having the binding specificity of a selected murine antibody(e.g., 16G7) can also be produced using a variant of the phage displaymethod. See Winter, WO 92/20791. This method is particularly suitablefor producing human antibodies. In this method, either the heavy orlight chain variable region of the selected murine antibody is used as astarting material. If, for example, a light chain variable region isselected as the starting material, a phage library is constructed inwhich members display the same light chain variable region (i.e., themurine starting material) and a different heavy chain variable region.The heavy chain variable regions can for example be obtained from alibrary of rearranged human heavy chain variable regions. A phageshowing strong specific binding for tau or a fragment thereof (e.g., atleast 10⁸ and preferably at least 10⁹ M⁻¹) is selected. The heavy chainvariable region from this phage then serves as a starting material forconstructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions can be obtained forexample from a library of rearranged human variable light chain regions.Again, phage showing strong specific binding for tau are selected. Theresulting antibodies usually have the same or similar epitopespecificity as the murine starting material.

Kabat/Chothia Composite CDRs of the heavy chain of 16G7 are designatedSEQ ID NOs: 8, 9, and 10, respectively, and Kabat CDRs of the lightchain of 16G7 are designated SEQ ID NOs: 12, 13, and 14, respectively.

TABLE 2 16G7 CDRs as defined by Kabat, Chothia, Composite of Chothia andKabat, AbM, and Contact Composite of Loop Kabat Chothia Chothia & KabatAbM Contact L1 L24 - - - L34 L24 - - - L34 L24 - - - L34 L24 - - - L34L30 - - - L36 SEQ ID NO: 12 SEQ ID NO: 12 SEQ ID NO: 12 SEQ ID NO: 12SEQ ID NO: 43 L2 L50 - - - L56 L50 - - - L56 L50 - - - L56 L50 - - - L56L46 - - - L55 SEQ ID NO: 13 SEQ ID NO: 13 SEQ ID NO: 13 SEQ ID NO: 13SEQ ID NO: 44 L3 L89 - - - L97 L89 - - - L97 L89 - - - L97 L89 - - - L97L89 - - - L96 SEQ ID NO: 14 SEQ ID NO: 14v SEQ ID NO: 14 SEQ ID NO: 14SEQ ID NO: 45 H1 H31 - - - H35B H26 - - - H32 H26 - - - H35B H26 - - -H35B H30 - - - H35B SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 8 SEQ ID NO:8 SEQ ID NO: 46 H2 H50 - - - H65 H52 - - - H56 H50 - - - H65 H50 - - -H58 H47 - - - H58 SEQ ID NO: 9 SEQ ID NO: 41 SEQ ID NO: 9 SEQ ID NO: 42SEQ ID NO: 47 H3 H95 - - - H102 H95 - - - H102 H95 - - - H102 H95 - - -H102 H93 - - - H101 SEQ ID NO: 10 SEQ ID NO: 10 SEQ ID NO: 10 SEQ ID NO:10 SEQ ID NO: 48

Other antibodies can be obtained by mutagenesis of cDNA encoding theheavy and light chains of an exemplary antibody, such as 16G7.Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%,98%, or 99% identical to 16G7 in amino acid sequence of the mature heavyand/or light chain variable regions and maintain its functionalproperties, and/or which differ from the respective antibody by a smallnumber of functionally inconsequential amino acid substitutions (e.g.,conservative substitutions), deletions, or insertions are also includedin the invention. Monoclonal antibodies having at least one or all sixCDR(s) as defined by any conventional definition, but preferably Kabat,that are 90%, 95%, 99% or 100% identical to corresponding CDRs of 16G7are also included.

The invention also provides antibodies having some or all (e.g., 3, 4,5, and 6) CDRs entirely or substantially from 16G7. Such antibodies caninclude a heavy chain variable region that has at least two, and usuallyall three, CDRs entirely or substantially from the heavy chain variableregion of 16G7 and/or a light chain variable region having at least two,and usually all three, CDRs entirely or substantially from the lightchain variable region of 16G7. The antibodies can include both heavy andlight chains. A CDR is substantially from a corresponding 16G7 CDR whenit contains no more than 4, 3, 2, or 1 substitutions, insertions, ordeletions, except that CDR-H2 (when defined by Kabat) can have no morethan 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Suchantibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identity to 16G7 in the amino acid sequence of the mature heavy and/orlight chain variable regions and maintain their functional properties,and/or differ from 16G7 by a small number of functionallyinconsequential amino acid substitutions (e.g., conservativesubstitutions), deletions, or insertions.

Some antibodies identified by such assays can bind to monomeric,misfolded, aggregated, phosphorylated, or unphosphorylated forms of tauor otherwise. Likewise, some antibodies are immunoreactive onnon-pathological and pathological forms and conformations of tau.

B. Non-Human Antibodies

The production of other non-human antibodies, e.g., murine, guinea pig,primate, rabbit or rat, against tau or a fragment thereof (e.g., aminoacid residues 55-78, 60-75, 61-70, 55-69, or 64-78 of SEQ ID NO: 3,corresponding to amino acid residues 113-136, 118-133, 119-128, 113-127,or 122-136, respectively, of SEQ ID NO:1) can be accomplished by, forexample, immunizing the animal with tau or a fragment thereof. SeeHarlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988)(incorporated by reference for all purposes). Such an immunogen can beobtained from a natural source, by peptide synthesis, or by recombinantexpression. Optionally, the immunogen can be administered fused orotherwise complexed with a carrier protein. Optionally, the immunogencan be administered with an adjuvant. Several types of adjuvant can beused as described below. Complete Freund's adjuvant followed byincomplete adjuvant is preferred for immunization of laboratory animals.Rabbits or guinea pigs are typically used for making polyclonalantibodies. Mice are typically used for making monoclonal antibodies.Antibodies are screened for specific binding to tau or an epitope withintau (e.g., an epitope comprising one or more of amino acid residues55-78, 60-75, or 61-70 of SEQ ID NO:3 (corresponding to amino acidresidues 113-136, 118-133, or 119-128, respectively, of SEQ ID NO:1).Such screening can be accomplished by determining binding of an antibodyto a collection of tau variants, such as tau variants containing aminoacid residues 55-78, 60-75, 61-70, 55-69, or 64-78 of SEQ ID NO:3(corresponding to amino acid residues 113-136, 118-133, 119-128,113-127, or 122-136, respectively, of SEQ ID NO:1) or mutations withinthese residues, and determining which tau variants bind to the antibody.Binding can be assessed, for example, by Western blot, FACS or ELISA.

C. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which CDRsfrom a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No.6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. Thus, a humanized antibody is an antibody having at leastthree, four, five or all CDRs entirely or substantially from a donorantibody and variable region framework sequences and constant regions,if present, entirely or substantially from human antibody sequences.Similarly a humanized heavy chain has at least one, two and usually allthree CDRs entirely or substantially from a donor antibody heavy chain,and a heavy chain variable region framework sequence and heavy chainconstant region, if present, substantially from human heavy chainvariable region framework and constant region sequences. Similarly ahumanized light chain has at least one, two and usually all three CDRsentirely or substantially from a donor antibody light chain, and a lightchain variable region framework sequence and light chain constantregion, if present, substantially from human light chain variable regionframework and constant region sequences. Other than nanobodies and dAbs,a humanized antibody comprises a humanized heavy chain and a humanizedlight chain. A CDR in a humanized antibody is substantially from acorresponding CDR in a non-human antibody when at least 85%, 90%, 95% or100% of corresponding residues (as defined by any conventionaldefinition but preferably defined by Kabat) are identical between therespective CDRs. The variable region framework sequences of an antibodychain or the constant region of an antibody chain are substantially froma human variable region framework sequence or human constant regionrespectively when at least 85%, 90%, 95% or 100% of correspondingresidues defined by Kabat are identical. To be classified as humanizedunder the 2014 World Health Organization (WHO) Internationalnon-proprietary names (INN) definition of humanized antibodies, anantibody must have at least 85% identity to human germline antibodysequences (i.e., prior to somatic hypermutation). Mixed antibodies areantibodies for which one antibody chain (e.g., heavy chain) meets thethreshold but the other chain (e.g., light chain) does not meet thethreshold. An antibody is classified as chimeric if neither chain meetsthe threshold, even though the variable framework regions for bothchains were substantially human with some murine backmutations. See,Jones et al. (2016) The INNs and outs of antibody nonproprietary names,mAbs 8:1, 1-9, DOI: 10.1080/19420862.2015.1114320. See also “WHO-INN:International nonproprietary names (INN) for biological andbiotechnological substances (a review)” (Internet) 2014. Available from:http://www.whoint/medicines/services/inn/BioRev2014.pdf), incorporatedherein by reference. For the avoidance of doubt, the term ³humanized² asused herein is not intended to be limited to the 2014 WHO INN definitionof humanized antibodies. Some of the humanized antibodies providedherein have at least 85% sequence identity to human germline sequencesand some of the humanized antibodies provided herein have less than 85%sequence identity to human germline sequences. Some of the heavy chainsof the humanized antibodies provided herein have from about 60% to 100%sequence identity to human germ line sequences, such as, for example, inthe range of about 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89%.Some heavy chains fall below the 2014 WHO INN definition and have, forexample, about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, or 84% sequence identity tohuman germ line sequences, while other heavy chains meet the 2014 WHOINN definition and have about 85%, 86%, 87%, 88%, 89% or greatersequence identity to human germ line sequences. Some of the light chainsof the humanized antibodies provided herein have from about 60% to 100%sequence identity to human germ line sequences, such as, for example, inthe range of about 80% to 84% or 85% to 89%. Some light chains fallbelow the 2014 WHO INN definition and have, for example, about 81%, 82%,83% or 84% sequence identity to human germ line sequences, while otherlight chains meet the 2014 WHO INN definition and have about 85%, 86%,87%, 88%, 89% or greater sequence identity to human germ line sequences.Some humanized antibodies provided herein that are “chimeric” under the2014 WHO INN definition have heavy chains with less than 85% identity tohuman germ line sequences paired with light chains having less than 85%identity to human germ line sequences. Some humanized antibodiesprovided herein are “mixed” under the 2014 WHO INN definition, forexample, having a heavy chain with at least 85% sequence identity tohuman germ line sequences paired with a light chain having less than 85%sequence identity to human germ line sequences, or vice versa. Somehumanized antibodies provided herein meet the 2014 WHO INN definition of“humanized” and have a heavy chain with at least 85% sequence identityto human germ line sequences paired with a light chain having at least85% sequence identity to human germ line sequences. Exemplary antibodiesthat meet the 2014 WHO INN definition of “humanized” include antibodieshaving a mature light chain with the amino acid sequence of SEQ ID NO:29paired with a mature heavy chain sequence having an amino acid sequenceof SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27. Additionalhumanized antibodies of the invention include antibodies having a matureheavy chain having an amino acid sequence of any of SEQ ID NOs:15-27paired with a mature light chain having an amino acid sequence of any ofSEQ ID NOs:28-30.

Although humanized antibodies often incorporate all six CDRs (defined byany conventional definition but preferably as defined by Kabat) from amouse antibody, they can also be made with less than all CDRs (e.g., atleast 3, 4, or 5 CDRs) from a mouse antibody (e.g., Pascalis et al., J.Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol., 320: 415-428,2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al,J. Immunol., 164:1432-1441, 2000).

In some antibodies only part of the CDRs, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting antigen andnot in the SDRs can be identified based on previous studies (for exampleresidues H60-H65 in CDR H2 are often not required), from regions ofKabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol.Biol. 196:901, 1987), by molecular modeling and/or empirically, or asdescribed in Gonzales et al., Mol. Immunol. 41: 863, 2004. In suchhumanized antibodies at positions in which one or more donor CDRresidues is absent or in which an entire donor CDR is omitted, the aminoacid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity and/or for meeting theWHO INN definition of “humanized”. However, substitutions can also causechanges of affinity, and significant reductions in affinity arepreferably avoided. Positions for substitution within CDRs and aminoacids to substitute can also be selected empirically.

The human acceptor antibody sequences can optionally be selected fromamong the many known human antibody sequences to provide a high degreeof sequence identity (e.g., 65-85% identity) between a human acceptorsequence variable region frameworks and corresponding variable regionframeworks of a donor antibody chain.

An example of an acceptor sequence for the heavy chain is the humanmature heavy chain variable region with NCBI accession code AAA18265.1(SEQ ID NO:31). An example of an acceptor sequence for the heavy chainis the human mature heavy chain variable region with NCBI accession codeADW08092.1 (SEQ ID NO:32). An example of an acceptor sequence for theheavy chain is the human mature heavy chain variable region with NCBIaccession code IMGT# IGHV1-2*02 (SEQ ID NO:33). IMGT# IGHV1-2*02includes CDRs CDR-H1 and CDR-H2 having the same canonical form as mouse16G7 heavy chain and is a member of Kabat human chain heavy subgroup 1.An example of an acceptor sequence for the light chain is the humanmature light chain variable region with NCBI accession code AAB24404.1(SEQ ID NO: 34). An example of an acceptor sequence for the light chainis the human mature light chain variable region with NCBI accession codeAEK69364.1 (SEQ ID NO: 35). An example of an acceptor sequence for thelight chain is the human mature light chain variable region with NCBIaccession code IMGT#IGKV4-1*01 (SEQ ID NO: 36). IMGT#IGKV4-1*01 has thesame canonical classes for CDR-L1, CDR-L2 and CDR-L3 as does mouse 16G7,and belongs to human kappa subgroup 1.

If more than one human acceptor antibody sequence is selected, acomposite or hybrid of those acceptors can be used, and the amino acidsused at different positions in the humanized light chain and heavy chainvariable regions can be taken from any of the human acceptor antibodysequences used.

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly;    -   (2) is adjacent to a CDR region or within a CDR as defined by        Chothia but not Kabat;    -   (3) otherwise interacts with a CDR region (e.g., is within about        6 Å of a CDR region), (e.g., identified by modeling the light or        heavy chain on the solved structure of a homologous known        immunoglobulin chain); or    -   (4) is a residue participating in the VL-VH interface.

The invention provides humanized forms of the murine 16G7 antibodyincluding 13 exemplified humanized mature heavy chain variable regions:hu16G7VHv1 (SEQ ID NO:15), hu16G7VHv2) (SEQ ID NO:16), hu16G7VHv2a (SEQID NO:17), 16G7VHv2aB7 (SEQ ID NO:18), hu16G7VHv2b (SEQ ID NO:19),hu16G7VHv2bH7 (SEQ ID NO:20), hu16G7VHv3 (SEQ ID NO:21), hu16G7VHv4 (SEQID NO:22), hu16G7VHv5V (SEQ ID NO:23), hu16G7VHv5VR (SEQ ID NO:24),hu16G7VHv6a (SEQ ID NO:25), hu16G7VHv6b (SEQ ID NO:26 hu16G7VHv7 (SEQ IDNO:27). and 3 exemplified human mature light chain variable regions:hu16G7VLv1 (SEQ ID NO:28). hu16G7VLv2 (SEQ ID NO:29), and hu16G7VLv3(SEQ ID NO:30).

In an embodiment, humanized sequences are generated using a two-stagePCR protocol that allows introduction of multiple mutations, deletions,and insertions using QuikChange site-directed mutagenesis [Wang, W. andMalcolm, B. A. (1999) BioTechniques 26:680-682)].

Framework residues from classes (1) through (3) as defined by Queen,U.S. Pat. No. 5,530,101, are sometimes alternately referred to ascanonical and vernier residues. Framework residues that help define theconformation of a CDR loop are sometimes referred to as canonicalresidues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton &Martin, J. Mol. Biol. 263:800-815 (1996)). Framework residues thatsupport antigen-binding loop conformations and play a role infine-tuning the fit of an antibody to antigen are sometimes referred toas vernier residues (Foote & Winter, J. Mol. Biol 224:487-499 (1992)).

Other framework residues that are candidates for substitution areresidues creating a potential glycosylation site. Still other candidatesfor substitution are acceptor human framework amino acids that areunusual for a human immunoglobulin at that position. These amino acidscan be substituted with amino acids from the equivalent position of themouse donor antibody or from the equivalent positions of more typicalhuman immunoglobulins.

Other framework residues that are candidates for substitution areN-terminal glutamine residues (Q) that may be replaced with glutamicacid (E) to minimize potential for pyroglutamate conversion [Y. DianaLiu, et al., 2011, J. Biol. Chem., 286: 11211-11217]. Glutamic acid (E)conversion to pyroglutamate (pE) occurs more slowly than from glutamine(Q). Because of the loss of a primary amine in the glutamine to pEconversion, antibodies become more acidic. Incomplete conversionproduces heterogeneity in the antibody that can be observed as multiplepeaks using charge-based analytical methods. Heterogeneity differencesmay indicate a lack of process control. Exemplary humanized antibodieswith N-terminal glutamine to glutamate substitutions are SEQ ID NO:15(hu16G7VHv1), SEQ ID NO:16 (hu16G7VHv2), SEQ ID NO:17 (hu16G7VHv2a), SEQID NO:18 (16G7VHv2aB7), SEQ ID NO:19 (hu16G7VHv2b), SEQ ID NO:20(hu16G7VHv2bH7), SEQ ID NO:21 (hu16G7VHv3), SEQ ID NO:22 (hu16G7VHv4),SEQ ID NO:23 (hu16G7VHv5V), SEQ ID NO:24 (hu16G7VHv5VR), and SEQ IDNO:27 (hu16G7VHv7).

Exemplary humanized antibodies are humanized forms of the mouse 16G7,designated Hu16G7.

The mouse antibody 16G7 comprises mature heavy and light chain variableregions having amino acid sequences comprising SEQ ID NO: 7 and SEQ IDNO:11, respectively. The invention provides 13 exemplified humanizedmature heavy chain variable regions: hu16G7VHv1, hu16G7VHv2),hu16G7VHv2a, 16G7VHv2aB7, hu16G7VHv2b, hu16G7VHv2bH7, hu16G7VHv3,hu16G7VHv4, hu16G7VHv5V, hu16G7VHv5VR, hu16G7VHv6a, hu16G7VHv6b, andhu16G7VHv7. The invention further provides 3 exemplified human maturelight chain variable regions: hu16G7VLv1. hu16G7VLv2, and hu16G7VLv3.FIGS. 2 and 3 show alignments of the heavy chain variable region andlight chain variable region, respectively, of murine 16G7 and varioushumanized antibodies.

For reasons such as possible influence on CDR conformation and/orbinding to antigen, mediating interaction between heavy and lightchains, interaction with the constant region, being a site for desiredor undesired post-translational modification, being an unusual residuefor its position in a human variable region sequence and thereforepotentially immunogenic, getting aggregation potential, and otherreasons, the following 33 variable region framework positions wereconsidered as candidates for substitutions in the 3 exemplified humanmature light chain variable regions and the 13 exemplified human matureheavy chain variable regions, as further specified in the examples: L4(M4L), L9 (D9S), L15 (115V), L18 (R18K), L19 (A19V), L21 (I21M), L22(N22S), L43 (P43S), L48 (I48M), H1 (Q1E), H5 (V5Q), H7 (S7P), H9 (A9S),H10 (E10V), H11 (V11L), H12 (K12V), H13 (K13R), H20 (V20L), H37 (V37A),H38 (R38K), H40 (A40R), H48 (M48I), H66 (R66K), H67 (V67K), H69 (M69L),H71 (R71V), H73 (T73I), H75 (I75S or I75A), H80 (M80V), H82a (S82a-T),H82b (R82b-S), H83 (R83T), H85 (D85E). The following 3 variable regionCDR positions were considered as candidates for substitutions in the 13exemplified human mature heavy chain variable regions, as furtherspecified in the examples: H31 (S31G), H60 (N60A), and H64 (K64Q).

Here, as elsewhere, the first-mentioned residue is the residue of ahumanized antibody formed by grafting Kabat CDRs or a compositeChothia-Kabat CDR in the case of CDR-H1 into a human acceptor framework,and the second-mentioned residue is a residue being considered forreplacing such residue. Thus, within variable region frameworks, thefirst mentioned residue is human, and within CDRs, the first mentionedresidue is mouse.

Exemplified antibodies include any permutations or combinations of theexemplified mature heavy and light chain variable regions e.g.,VHv1/VLv1, VHv1/VLv2, VHv1/VLv3, VHv2/VLv1, VHv2/VLv2, VHv2/VLv3,VHv2a/VLv1, VHv2a/VLv2, VHv2a/VLv3, VHv2aB7/VLv1, VHv2aB7/VLv2,VHv2aB7/VLv3, VHv2b/VLv1, VHv2b/VLv2, VHv2b/VLv3, VHv2bH7/VLv1,VHv2bH7/VLv2, VHv2bH7/VLv3, VHv3/VLv1, VHv3/VLv2, VHv3/VLv3, VHv4/VLv1,VHv4/VLv2, VHv4/VLv3, VHv5/VLv1, VHv5/VLv2, VHv5/VLv3, VHv5VR/VLv1,VHv5VR/VLv2, VHv5VR/VLv3, VHv6a/VLv1, VHv6a/VLv2, VHv6a/VLv3,VHv6b/VLv1, VHv6b/VLv2, VHv6b/VLv3, VHv7/VLv1, VHv7/VLv2, or VHv7/VLv3.

The invention provides variants of the 16G7 humanized antibody in whichthe humanized mature heavy chain variable region shows at least 90%,95%, 96%, 97%, 98%, or 99% identity to hu16G7VHv1, hu16G7VHv2,hu16G7VHv2a, 16G7VHv2aB7, hu16G7VHv2b, hu16G7VHv2bH7, hu16G7VHv3,hu16G7VHv4, hu16G7VHv5V, hu16G7VHv5VR, hu16G7VHv6a, hu16G7VHv6b, andhu16G7VHv7 (SEQ ID NOs: 15-27) and the humanized mature light chainvariable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identityto hu16G7VLv1. hu16G7VLv2, and hu16G7VLv3 (SEQ ID NO: 28-30). In somesuch antibodies at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, or all 36 of the backmutations or other mutations found inSEQ ID NO:15-27 and SEQ ID NO:28-30 are retained.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H1 is occupied by E, H5 is occupied by Q, H7 is occupied by P, H9 isoccupied by S, H10 is occupied by V, H11 is occupied L, H12 is occupiedby V, H13 is occupied by R, H20 is occupied by L, H40 is occupied by R,H48 is occupied by I, H66 is occupied by K, H67 is occupied by A, H69 isoccupied by L, H71 is occupied by V, H73 is occupied by I, H75 isoccupied by S, H80 is occupied by V, H82a is occupied by T, H82b isoccupied by S, H83 is occupied by T, and H85 is occupied by E. In somehumanized 16G7 antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13,H20, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83, andH85 in the VH region are occupied by, E, Q, P, S, V, L, V, R, L, R, I,K, A, L, V, I, S, V, T, S, T, and E, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H1 is occupied by E, H5 is occupied by Q, H7 is occupied by P, H9 isoccupied by S, H10 is occupied by V, H11 is occupied by L, H12 isoccupied by V, H13 is occupied by R, H20 is occupied by L, H48 isoccupied by I, H69 is occupied by L, H71 is occupied by V, H82a isoccupied by T, H82b is occupied by S, H83 is occupied by T, and H85 isoccupied by E. In some humanized 16G7 antibodies, positions H1, H5, H7,H9, H10, H11, H12, H13, H20, H48, H69, H71, H82a, H82b, H83, and H85 inthe VH region are occupied by E, Q, P, S, V, L, V, R, L, I, L, V, T, S,T, and E, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H1 is occupied by E, H5 is occupied by Q, H11 is occupied by L, H12 isoccupied by V, H20 is occupied by L, H48 is occupied by I, H69 isoccupied by L, H71 is occupied by V, H82a is occupied by T, H82b isoccupied by S, H83 is occupied by T, and H85 is occupied by E. In somehumanized 16G7 antibodies, positions H1, H5, H11, H12, H20, H48, H69,H71, H82a, H82b, H83, and H85. in the VH region are occupied by E, Q, L,V, L I, L, V, T, S, T, and E, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H1 is occupied by E, H12 is occupied by V, H31 is occupied by G, H40 isoccupied by R, H48 is occupied by I, H60 is occupied by A, H69 isoccupied by L, H73 is occupied by I, and H83 is occupied by T. In somehumanized 16G7 antibodies, positions H1, H12, H31, H40, H48, H60, H69,H73, and H83 in the VH region are occupied by are occupied by E, V, G,R, I, A, L, I, and T, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H1 is occupied by E, H12 is occupied by V, H40 is occupied by R, H48 isoccupied by I, H69 is occupied by L, H73 is occupied by I, and H83 isoccupied by T. In some humanized 16G7 antibodies, positions H1, H12,H40, H48, H69, H73, and H83 in the VH region are occupied by areoccupied by E, V, R, I, L, I, and T, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H1 is occupied by E, H12 is occupied by V, H40 is occupied by R, H48 isoccupied by I, H66 is occupied K, H67 is occupied by A, H69 is occupiedby L, H71 is occupied by V, H73 is occupied by I, and H83 is occupied byT. In some humanized 16G7 antibodies, positions H1, H12, H40, H48, H66,H67, H69, H71, H73, and H83 in the VH region are occupied by areoccupied by E, V, R, I, K, A, L, V, I, and T, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H1 is occupied by E, H12 is occupied by V, H40 is occupied by R, H48 isoccupied by I, H69 is occupied by L, H73 is occupied by I, and H83 isoccupied by T. In some humanized 16G7 antibodies, positions H1, H12,H40, H48, H69, H73, and H83 in the VH region are occupied by areoccupied by E, V, R, I, L, I, and T, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H7 is occupied by P, H71 is occupied by V, and H73 is occupied by I. Insome humanized 16G7 antibodies, positions H7, H71, and H73 in the VHregion are occupied by are occupied by P, V, and I, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H31 is occupied by G and H60 is occupied by A. In some humanized 16G7antibodies, positions H31 and H60 in the VH region are occupied by G andA, respectively.

In some humanized 16G7 antibodies, positions H1, H12, H31, H40, H48,H60, H66, H67, H69, H71, H73, and H83 in the VH region are occupied byQ, V, G, R, I, A, K, A, L, V, I, and T, respectively.

In some humanized 16G7 antibodies, positions H1, H7, H71, and H73 in theVH region are occupied by Q, P, V and I, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H1 is occupied by Q or E, H5 is occupied by V or Q, H7 is occupied by Sor P, H9 is occupied by A or S, H10 is occupied by E or V, H11 isoccupied V or L, H12 is occupied by K or V, H13 is occupied by K or R,H20 is occupied by V or L, H31 is occupied by G or S, H37 is occupied byV or A, H 38 is occupied by R or K, H40 is occupied by A or R, H48 isoccupied by M or I, H60 is occupied by A or N, H64 is occupied by Q orK, H66 is occupied by R or K, H67 is occupied by V or A, H69 is occupiedby M or L, H71 is occupied by R or V, H73 is occupied by T or I, H75 isoccupied by I, S, or A, H80 is occupied by M or V, H82a is occupied by Sor T, H82b is occupied by R or S, H83 is occupied by R or T, and H85 isoccupied by D or E.

In some humanized 16G7 antibodies, positions H1, H5, H7, H9, H10, H11,H12, H13, H20, H37, H38, H40, H48, H66, H67, H69, H71, H73, H75, H80,H82a, H82b, H83, and H85 in the VH region are occupied by E, Q, P, S, V,L, V, R, L, A, K, R, I, K, A, L, V, I, S, V, T, S, T, and E,respectively, as in hu16G7VHv1. In some humanized 16G7 antibodies,positions H1, H5, H11, H12, H20, H48, H69, H71, H75, H82a, H82b, H83,and H85 in the VH region are occupied by E, Q, L, V, L, I, L, V, A, T,S, T, and E, respectively, as in hu16G7VHv2. In some humanized 16G7antibodies, positions H1, H12, H40, H48, H66, H67, H69, H71, H73, andH83 in the VH region are occupied by E, V, R, I, K, A, L, V, I, and T,respectively, as in hu16G7VHv2a. In some humanized 16G7 antibodies,positions H1, H12, H31, H40, H48, H60, H66, H67, H69, H71, H73, and H83in the VH region are occupied by E, V, G, R, I, A, K, A, L, V, I, and T,respectively, as in hu16G7VHv2aB7. In some humanized 16G7 antibodies,positions H1, H12, H40, H48, H69, H73, and H83 in the VH region areoccupied by E, V, R, I, L, I, and T, respectively, as in hu16G7VHv2b. Insome humanized 16G7 antibodies, positions H1, H12, H31, H40, H48, H60,H64, H69, H73, and H83 in the VH region are occupied by, E, V, G, R, I,A, Q, L, I, and T, respectively, as in hu16G7VHv2bH7. In some humanized16G7 antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H48,H69, H71, H75, H82a, H82b, H83, and H85 in the VH region are occupied byE, Q, P, S, V, L, V, R, L, I, L, V, A, T, S, T, and E, respectively, asin hu16G7VHv3. In some humanized 16G7 antibodies, positions H1, H5, H11,H12, H20, H48, H66, H67, H69, H71, H73, H75, H82a, H82b, H83, and H85 inthe VH region are occupied by, E, Q, L, V, L, I, K, A, L, V, I, S, T, S,T, and E, respectively, as in hu16G7VHv4. In some humanized 16G7antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H38, H40,H48, H66, H67, H69, H71, H73I, H75, H80, H82a, H82b, H83, and H85 in theVH region are occupied by E, Q, P, S, V, L, V, R, L, K, R, I, K, A, L,V, I, S, V, T, S, T, and E, respectively, as in hu16G7VHv5V. In somehumanized 16G7 antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13,H20, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83, andH85 in the VH region are occupied by Q, P, S, V, L, V, R, L, R, I, K, A,L, V, I, S, V, T, S, T, and E, respectively, as in hu16G7VHv5VR. In somehumanized 16G7 antibodies, positions H7, H71, and H73 in the VH regionare occupied by P, V, and I, respectively, as in hu16G7VHv6a. In somehumanized 16G7 antibodies, position H71 in the VH region is occupied byV, as in hu16G7VHv6b. In some humanized 16G7 antibodies, positions H1,H7, H71, and H73 in the VH region are occupied by E, P, V, and I,respectively, as in hu16G7VHv7.

In some humanized 16G7 antibodies, the variable heavy chain has ≥85%identity to human sequence. In some humanized 16G7 antibodies, thevariable light chain has ≥85% identity to human sequence. In somehumanized 16G7 antibodies, each of the variable heavy chain and variablelight chain has ≥85% identity to human germline sequence.

In some humanized 16G7 antibodies, the three heavy chain CDRs are asdefined by Kabat/Chothia Composite (SEQ ID NOs: 8, 9, and 10) and thethree light chain CDRs are as defined by Kabat/Chothia Composite (SEQ IDNOs: 12, 13, and 14); provided that position H31 is occupied by S or G,position H60 is occupied by N or A and position 64 is occupied by K orQ. In some humanized 16G7 antibodies, CDR-H1 has an amino acid sequencecomprising SEQ ID NO: 49. In some humanized 16G7 antibodies, CDR-H2 hasan amino acid sequence comprising SEQ ID NO: 50. In some humanized 16G7antibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO: 51.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:L4 is occupied by L, L9 is occupied by S, L15 is occupied by V, L22 isoccupied by S, and L43 is occupied by S. In some humanized 16G7antibodies, positions L4, L9, L15, L22, and L43 in the VL region areoccupied by L, S, V, S, and S, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:L4 is occupied by L, L9 is occupied by S, L15 is occupied by V, L18 isoccupied by K, L19 is occupied by V, L21 is occupied by M, L22 isoccupied by S, and L43 is occupied by S. In some humanized 16G7antibodies, positions L4, L9, L15, L18, L19, L21, L22, and L43 in the VLregion are occupied by L, S, V, K, V, M, S, and S, respectively.

In some humanized 16G7 antibodies, at least one of the followingpositions in the VL region is occupied by the amino acid as specified:L4 is occupied by M or L, L9 is occupied by D or S, L15 is occupied by Lor V, L18 is occupied by R or K, L19 is occupied by A or V, L21 isoccupied by I or M, L22 is occupied by N or S, L43 is occupied by P orS, and L48 is occupied by I or M.

In some humanized 16G7 antibodies, positions L4, L9, L15, L18, L19, L21,L22, and L43 in the VL region are occupied by L, S, V, K, V, M, S, andS, respectively, as in hu16G7VLv1. In some humanized 16G7 antibodies,positions L4, L9, L15, L22, and L43 in the VL region are occupied by L,S, V, S, and S, respectively, as in hu16G7VLv2. In some humanized 16G7antibodies, positions L4, L9, L15, L18, L19, L21, L22, and L43 in the VLregion are occupied by L, S, V, K, V, M, S, and S, respectively, as inhu16G7VLv3.

In some humanized 16G7 antibodies, the variable heavy chain has 85%identity to human sequence. In some humanized 16G7 antibodies, thevariable light chain has 85% identity to human sequence. In somehumanized 16G7 antibodies, each of the variable heavy chain and variablelight chain has 85% identity to human germline sequence. In somehumanized 16G7 antibodies, the three heavy chain CDRs are as defined byKabat/Chothia Composite (SEQ ID NOs: 8, 9, and 10) and the three lightchain CDRs are as defined by Kabat/Chothia Composite (SEQ ID NOs: 12,13, and 14); provided that position H31 is occupied by S or G, positionH60 is occupied by N or A and position 64 is occupied by K or Q. In somehumanized 16G7 antibodies, CDR-H1 has an amino acid sequence comprisingSEQ ID NO: 49. In some humanized 16G7 antibodies, CDR-H2 has an aminoacid sequence comprising SEQ ID NO: 50. In some humanized 16G7antibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO: 51.

The CDR regions of such humanized antibodies can be identical orsubstantially identical to the CDR regions of 16G7. The CDR regions canbe defined by any conventional definition (e.g., Chothia, or compositeof Chothia and Kabat) but are preferably as defined by Kabat.

Variable regions framework positions are in accordance with Kabatnumbering unless otherwise stated. Other such variants typically differfrom the sequences of the exemplified Hu16G7 heavy and light chains by asmall number (e.g., typically no more than 1, 2, 3, 5, 10, or 15) ofreplacements, deletions or insertions. Such differences are usually inthe framework but can also occur in the CDRs.

A possibility for additional variation in humanized 16G7 variants isadditional backmutations in the variable region frameworks. Many of theframework residues not in contact with the CDRs in the humanized mAb canaccommodate substitutions of amino acids from the correspondingpositions of the donor mouse mAb or other mouse or human antibodies, andeven many potential CDR-contact residues are also amenable tosubstitution. Even amino acids within the CDRs may be altered, forexample, with residues found at the corresponding position of the humanacceptor sequence used to supply variable region frameworks. Inaddition, alternate human acceptor sequences can be used, for example,for the heavy and/or light chain. If different acceptor sequences areused, one or more of the backmutations recommended above may not beperformed because the corresponding donor and acceptor residues arealready the same without backmutations.

Preferably, replacements or backmutations in humanized 16G7 variants(whether or not conservative) have no substantial effect on the bindingaffinity or potency of the humanized mAb, that is, its ability to bindto tau).

The humanized 16G7 antibodies are further characterized by their abilityto bind both phosphorylated and unphosphorylated tau andmisfolded/aggregated forms of tau.

D. Chimeric and Veneered Antibodies

The invention further provides chimeric and veneered forms of non-humanantibodies, particularly the 16G7 antibodies of the examples.

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence. In anembodiment, a chimeric 16G7 antibody has a heavy chain amino acidsequence of SEQ ID NO: 54 and a light chain amino acid sequence of SEQID NO:55.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions. Veneered forms of the 16G7 antibody are included in theinvention.

E. Human Antibodies

Human antibodies against tau or a fragment thereof (e.g., amino acidresidues 55-78, 60-75, 61-70, 55-69, or 64-78 of SEQ ID NO: 3,corresponding to amino acid residues 113-136, 118-133, 119-128, 113-127,or 122-136, respectively, of SEQ ID NO:1) are provided by a variety oftechniques described below. Some human antibodies are selected bycompetitive binding experiments, by the phage display method of Winter,above, or otherwise, to have the same epitope specificity as aparticular mouse antibody, such as one of the mouse monoclonalantibodies described in the examples. Human antibodies can also bescreened for a particular epitope specificity by using only a fragmentof tau, such as a tau fragment containing only, amino acid residues55-78, 60-75, 61-70, 55-69, or 64-78 of SEQ ID NO: 3 (corresponding toamino acid residues 113-136, 118-133, 119-128, 113-127, or 122-136,respectively, of SEQ ID NO:1), as the target antigen, and/or byscreening antibodies against a collection of tau variants, such as tauvariants containing various mutations within amino acid residues 55-78,60-75, 61-70, 55-69, or 64-78 of SEQ ID NO: 3 (corresponding to aminoacid residues 113-136, 118-133, 119-128, 113-127, or 122-136,respectively, of SEQ ID NO:1).

Methods for producing human antibodies include the trioma method ofOestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No.4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, use oftransgenic mice including human immunoglobulin genes (see, e.g., Lonberget al., WO93/12227 (1993); U.S. Pat. Nos. 5,877,397; 5,874,299;5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126;5,569,825; 5,545,806; Neuberger, Nat. Biotechnol. 14:826 (1996); andKucherlapati, WO 91/10741 (1991)) phage display methods (see, e.g.,Dower et al., WO 91/17271; McCafferty et al., WO 92/01047; U.S. Pat.Nos. 5,877,218; 5,871,907; 5,858,657; 5,837,242; 5,733,743; and5,565,332); and methods described in WO 2008/081008 (e.g., immortalizingmemory B cells isolated from humans, e.g., with EBV, screening fordesired properties, and cloning and expressing recombinant forms).

F. Selection of Constant Region

The heavy and light chain variable regions of chimeric, veneered orhumanized antibodies can be linked to at least a portion of a humanconstant region. The choice of constant region depends, in part, whetherantibody-dependent cell-mediated cytotoxicity, antibody dependentcellular phagocytosis and/or complement dependent cytotoxicity aredesired. For example, human isotypes IgG1 and IgG3 havecomplement-dependent cytotoxicity and human isotypes IgG2 and IgG4 donot. Human IgG1 and IgG3 also induce stronger cell mediated effectorfunctions than human IgG2 and IgG4. Light chain constant regions can belambda or kappa. Numbering conventions for constant regions include EUnumbering (Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969)), Kabat numbering (Kabat, Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md., 1991, IMGTunique numbering (Lefranc M.-P. et al., IMGT unique numbering forimmunoglobulin and T cell receptor constant domains and Ig superfamilyC-like domains, Dev. Comp. Immunol., 29, 185-203 (2005), and IMGT exonnumbering (Lefranc, supra).

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering is used in this paragraphfor the constant region) for increasing the half-life of an antibody.Substitution at any or all of positions 234, 235, 236 and/or 237 reduceaffinity for Fcγ receptors, particularly FcγRI receptor (see, e.g., U.S.Pat. No. 6,624,821). An alanine substitution at positions 234, 235, and237 of human IgG1 can be used for reducing effector functions. Someantibodies have alanine substitution at positions 234, 235 and 237 ofhuman IgG1 for reducing effector functions. Optionally, positions 234,236 and/or 237 in human IgG2 are substituted with alanine and position235 with glutamine (see, e.g., U.S. Pat. No. 5,624,821). In someantibodies, a mutation at one or more of positions 241, 264, 265, 270,296, 297, 322, 329, and 331 by EU numbering of human IgG1 is used. Insome antibodies, a mutation at one or more of positions 318, 320, and322 by EU numbering of human IgG1 is used. In some antibodies, positions234 and/or 235 are substituted with alanine and/or position 329 issubstituted with glycine. In some antibodies, positions 234 and 235 aresubstituted with alanine. In some antibodies, the isotype is human IgG2or IgG4.

Antibodies can be expressed as tetramers containing two light and twoheavy chains, as separate heavy chains, light chains, as Fab, Fab′,F(ab′)2, and Fv, or as single chain antibodies in which heavy and lightchain mature variable domains are linked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype bind to a non-polymorphic region of a one or more otherisotypes. Thus, for example, another heavy chain constant region is ofIgG1 Glm3 with or without the C-terminal lysine. Reference to a humanconstant region includes a constant region with any natural allotype orany permutation of residues occupying positions in natural allotypes.

G. Expression of Recombinant Antibodies

A number of methods are known for producing chimeric and humanizedantibodies using an antibody-expressing cell line (e.g., hybridoma). Forexample, the immunoglobulin variable regions of antibodies can be clonedand sequenced using well known methods. In one method, the heavy chainvariable VH region is cloned by RT-PCR using mRNA prepared fromhybridoma cells. Consensus primers are employed to the VH region leaderpeptide encompassing the translation initiation codon as the 5′ primerand a g2b constant regions specific 3′ primer. Exemplary primers aredescribed in U.S. patent publication US 2005/0009150 by Schenk et al.(hereinafter “Schenk”). The sequences from multiple, independentlyderived clones can be compared to ensure no changes are introducedduring amplification. The sequence of the VH region can also bedetermined or confirmed by sequencing a VH fragment obtained by 5′ RACERT-PCR methodology and the 3′ g2b specific primer.

The light chain variable VL region can be cloned in an analogous manner.In one approach, a consensus primer set is designed for amplification ofVL regions using a 5′ primer designed to hybridize to the VL regionencompassing the translation initiation codon and a 3′ primer specificfor the Ck region downstream of the V-J joining region. In a secondapproach, 5′RACE RT-PCR methodology is employed to clone a VL encodingcDNA. Exemplary primers are described in Schenk, supra. The clonedsequences are then combined with sequences encoding human (or othernon-human species) constant regions.

In one approach, the heavy and light chain variable regions arere-engineered to encode splice donor sequences downstream of therespective VDJ or VJ junctions and are cloned into a mammalianexpression vector, such as pCMV-hγ1 for the heavy chain and pCMV-Mcl forthe light chain. These vectors encode human γ1 and Ck constant regionsas exonic fragments downstream of the inserted variable region cassette.Following sequence verification, the heavy chain and light chainexpression vectors can be co-transfected into CHO cells to producechimeric antibodies. Conditioned media is collected 48 hourspost-transfection and assayed by western blot analysis for antibodyproduction or ELISA for antigen binding. The chimeric antibodies arehumanized as described above.

Chimeric, veneered, humanized, and human antibodies are typicallyproduced by recombinant expression. Recombinant polynucleotideconstructs typically include an expression control sequence operablylinked to the coding sequences of antibody chains, including naturallyassociated or heterologous expression control elements, such as apromoter. The expression control sequences can be promoter systems invectors capable of transforming or transfecting eukaryotic orprokaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences and the collection andpurification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g., ampicillinresistance or hygromycin resistance, to permit detection of those cellstransformed with the desired DNA sequences.

E. coli is one prokaryotic host useful for expressing antibodies,particularly antibody fragments. Microbes, such as yeast, are alsouseful for expression. Saccharomyces is a yeast host with suitablevectors having expression control sequences, an origin of replication,termination sequences, and the like as desired. Typical promotersinclude 3-phosphoglycerate kinase and other glycolytic enzymes.Inducible yeast promoters include, among others, promoters from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

Mammalian cells can be used for expressing nucleotide segments encodingimmunoglobulins or fragments thereof. See Winnacker, From Genes toClones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped, and include CHO cell lines, various COS cell lines, HeLacells, HEK293 cells, L cells, and non-antibody-producing myelomasincluding Sp2/0 and NSO. The cells can be nonhuman. Expression vectorsfor these cells can include expression control sequences, such as anorigin of replication, a promoter, an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Expression control sequences caninclude promoters derived from endogenous genes, cytomegalovirus, SV40,adenovirus, bovine papillomavirus, and the like. See Co et al., J.Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. Nos. 5,741,957; 5,304,489; and 5,849,992). Suitable transgenesinclude coding sequences for light and/or heavy chains operably linkedwith a promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by methods depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics, or viral-based transfection can be used forother cellular hosts. Other methods used to transform mammalian cellsinclude the use of polybrene, protoplast fusion, liposomes,electroporation, and microinjection. For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected of FACS-based single-cell cloning to generatemonoclonal lines. Specific productivities above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, can be used. Antibodies produced by single cell clones can alsobe tested for turbidity, filtration properties, PAGE, IEF, UV scan,HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, andbinding assay, such as ELISA or Biacore. A selected clone can then bebanked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, HPLC purification,column chromatography, gel electrophoresis and the like (see generally,Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies can be employed,including codon optimization, selection of promoters, selection oftranscription elements, selection of terminators, serum-free single cellcloning, cell banking, use of selection markers for amplification ofcopy number, CHO terminator, or improvement of protein titers (see,e.g., U.S. Pat. Nos. 5,786,464; 6,114,148; 6,063,598; 7,569,339;WO2004/050884; WO2008/012142; WO2008/012142; WO2005/019442;WO2008/107388; WO2009/027471; and U.S. Pat. No. 5,888,809).

IV. Active Immunogens

An agent used for active immunization serves to induce in a patient thesame types of antibody described in connection with passive immunizationabove. Agents used for active immunization can be the same types ofimmunogens used for generating monoclonal antibodies in laboratoryanimals, e.g., a peptide of 3-15 or 3-12 or 5-12, or 5-8 contiguousamino acids from a region of tau corresponding to residues 55-78, 60-75,61-70, 55-69, or 64-78 of SEQ ID NO: 3 (corresponding to amino acidresidues 113-136, 118-133, 119-128, 113-127, or 122-136, respectively,of SEQ ID NO:1), such as, for example, a peptide including residues55-78, 60-75, 61-70, 55-69, or 64-78 of SEQ ID NO: 3 (corresponding toamino acid residues 113-136, 118-133, 119-128, 113-127, or 122-136,respectively, of SEQ ID NO:1). For inducing antibodies binding to thesame or overlapping epitope as 16G7, the epitope specificity of theseantibodies can be mapped (e.g., by testing binding to a series ofoverlapping peptides spanning tau). A fragment of tau consisting of orincluding or overlapping the epitope can then be used as an immunogen.Such fragments are typically used in unphosphorylated form.

The heterologous carrier and adjuvant, if used may be the same as usedfor generating monoclonal antibody, but may also be selected for betterpharmaceutical suitability for use in humans. Suitable carriers includeserum albumins, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from otherpathogenic bacteria, such as diphtheria (e.g., CRM197), E. coli,cholera, or H. pylori, or an attenuated toxin derivative. T cellepitopes are also suitable carrier molecules. Some conjugates can beformed by linking agents of the invention to an immunostimulatorypolymer molecule (e.g., tripalmitoyl-S-glycerine cysteine (Pam₃Cys),mannan (a mannose polymer), or glucan (a β 1→2 polymer)), cytokines(e.g., IL-1, IL-1 alpha and β peptides, IL-2, γ-INF, IL-10, GM-CSF), andchemokines (e.g., MIP1-α and β, and RANTES) Immunogens may be linked tothe carriers with or without spacers amino acids (e.g., gly-gly).Additional carriers include virus-like particles. Virus-like particles(VLPs), also called pseudovirions or virus-derived particles, representsubunit structures composed of multiple copies of a viral capsid and/orenvelope protein capable of self-assembly into VLPs of defined sphericalsymmetry in vivo. (Powilleit, et al., (2007) PLoS ONE 2(5):e415.)Alternatively, peptide immunogens can be linked to at least oneartificial T-cell epitope capable of binding a large proportion of MHCClass II molecules, such as the pan DR epitope (“PADRE”). PADRE isdescribed in U.S. Pat. No. 5,736,142, WO 95/07707, and Alexander J etal, Immunity, 1:751-761 (1994). Active immunogens can be presented inmultimeric form in which multiple copies of an immunogen and/or itscarrier are presented as a single covalent molecule.

Fragments are often administered with pharmaceutically acceptableadjuvants. The adjuvant increases the titer of induced antibodies and/orthe binding affinity of induced antibodies relative to the situation ifthe peptide were used alone. A variety of adjuvants can be used incombination with an immunogenic fragment of tau to elicit an immuneresponse. Preferred adjuvants augment the intrinsic response to animmunogen without causing conformational changes in the immunogen thataffect the qualitative form of the response. Preferred adjuvants includealuminum salts, such as aluminum hydroxide and aluminum phosphate, 3De-O-acylated monophosphoryl lipid A (MPL™) (see GB 2220211 (RIBIImmunoChem Research Inc., Hamilton, Mont., now part of Corixa).Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from thebark of the Quillaja Saponaria Molina tree found in South America (seeKensil et al., in Vaccine Design: The Subunit and Adjuvant Approach(eds. Powell & Newman, Plenum Press, N Y, 1995); U.S. Pat. No.5,057,540), (Aquila BioPharmaceuticals, Framingham, Mass.; nowAntigenics, Inc., New York, N.Y.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killedmycobacteria. Ribi adjuvants are oil-in-water emulsions. Ribi contains ametabolizable oil (squalene) emulsified with saline containing Tween 80.Ribi also contains refined mycobacterial products which act asimmunostimulants and bacterial monophosphoryl lipid A. Another adjuvantis CpG (WO 98/40100). Adjuvants can be administered as a component of atherapeutic composition with an active agent or can be administeredseparately, before, concurrently with, or after administration of thetherapeutic agent.

Analogs of natural fragments of tau that induce antibodies against taucan also be used. For example, one or more or all L-amino acids can besubstituted with D amino acids in such peptides. Also the order of aminoacids can be reversed (retro peptide). Optionally a peptide includes allD-amino acids in reverse order (retro-inverso peptide). Peptides andother compounds that do not necessarily have a significant amino acidsequence similarity with tau peptides but nevertheless serve as mimeticsof tau peptides and induce a similar immune response, Anti-idiotypicantibodies against monoclonal antibodies to tau as described above canalso be used. Such anti-Id antibodies mimic the antigen and generate animmune response to it (see Essential immunology, Roit ed BlackwellScientific Publications, Palo Alto, Calif. 6th ed., p. 181).

Peptides (and optionally a carrier fused to the peptide) can also beadministered in the form of a nucleic acid encoding the peptide andexpressed in situ in a patient. A nucleic acid segment encoding animmunogen is typically linked to regulatory elements, such as a promoterand enhancer that allow expression of the DNA segment in the intendedtarget cells of a patient. For expression in blood cells, as isdesirable for induction of an immune response, promoter and enhancerelements from light or heavy chain immunoglobulin genes or the CMV majorintermediate early promoter and enhancer are suitable to directexpression. The linked regulatory elements and coding sequences areoften cloned into a vector. Antibodies can also be administered in theform of nucleic acids encoding the antibody heavy and/or light chains.If both heavy and light chains are present, the chains are preferablylinked as a single chain antibody. Antibodies for passive administrationcan also be prepared e.g., by affinity chromatography from sera ofpatients treated with peptide immunogens.

The DNA can be delivered in naked form (i.e., without colloidal orencapsulating materials). Alternatively a number of viral vector systemscan be used including retroviral systems (see, e.g., Lawrie and Tumin,Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors {see,e.g., Bett et al, J. Virol. 67, 591 1 (1993)); adeno-associated virusvectors {see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), viralvectors from the pox family including vaccinia virus and the avian poxviruses, viral vectors from the alpha virus genus such as those derivedfrom Sindbis and Semliki Forest Viruses (see, e.g., Dubensky et al., J.Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (seeU.S. Pat. No. 5,643,576) and rhabdoviruses, such as vesicular stomatitisvirus (see WO 96/34625) and papillomaviruses (Ohe et al., Human GeneTherapy 6, 325-333 (1995); Woo et al, WO 94/12629 and Xiao & Brandsma,Nucleic Acids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833, and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), (see, e.g., McGee et al., J. Micro Encap.1996).

H. Antibody Screening Assays

Antibodies can be initially screened for the intended bindingspecificity as described above. Active immunogens can likewise bescreened for capacity to induce antibodies with such bindingspecificity. In this case, an active immunogen is used to immunize alaboratory animal and the resulting sera tested for the appropriatebinding specificity.

Antibodies having the desired binding specificity can then be tested incellular and animal models. The cells used for such screening arepreferentially neuronal cells. A cellular model of tau pathology hasbeen reported in which neuroblastoma cells are transfected with afour-repeat domain of tau, optionally with a mutation associated withtau pathology (e.g., delta K280, see Khlistunova, Current AlzheimerResearch 4, 544-546 (2007)). In another model, tau is induced in theneuroblastoma N2a cell line by the addition of doxycycline. The cellmodels enable one to study the toxicity of tau to cells in the solubleor aggregated state, the appearance of tau aggregates after switching ontau gene expression, the dissolution of tau aggregates after switchingthe gene expression off again, and the efficiency of antibodies ininhibiting formation of tau aggregates or disaggregating them.

Antibodies or active immunogens can also be screened in transgenicanimal models of diseases associated with tau. Such transgenic animalscan include a tau transgene (e.g., any of the human isoforms) andoptionally a human APP transgene among others, such as a kinase thatphosphorylates tau, ApoE, presenilin or alpha synuclein. Such transgenicanimals are disposed to develop at least one sign or symptom of adisease associated with tau.

An exemplary transgenic animal is the K3 line of mice (Itner et al.,Proc. Natl. Acad. Sci. USA 105(41):15997-6002 (2008)). These mice have ahuman tau transgene with a K 369 I mutation (the mutation is associatedwith Pick's disease) and a Thy 1.2 promoter. This model shows a rapidcourse of neurodegeneration, motor deficit and degeneration of afferentfibers and cerebellar granule cells. Another exemplary animal is theJNPL3 line of mice. These mice have a human tau transgene with a P301Lmutation (the mutation is associated with frontotemporal dementia) and aThy 1.2 promoter (Taconic, Germantown, N.Y., Lewis, et al., Nat Genet.25:402-405 (2000)). These mice have a more gradual course ofneurodegeneration. The mice develop neurofibrillary tangles in severalbrain regions and spinal cord, which is hereby incorporated by referencein its entirety). This is an excellent model to study the consequencesof tangle development and for screening therapy that may inhibit thegeneration of these aggregates. Another advantage of these animals isthe relatively early onset of pathology. In the homozygous line,behavioral abnormalities associated with tau pathology can be observedat least as early as 3 months, but the animals remain relatively healthyat least until 8 months of age. In other words, at 8 months, the animalsambulate, feed themselves, and can perform the behavioral taskssufficiently well to allow the treatment effect to be monitored. Activeimmunization of these mice for 6-13 months with—AI wI KLH-PHF-1generated titers of about 1,000 and showed fewer neurofibrillarytangles, less pSer422, and reduced weight loss relative to untreatedcontrol mice.

The activity of antibodies or active agents can be assessed by variouscriteria including reduction in amount of total tau or phosphorylatedtau, reduction in other pathological characteristics, such as amyloiddeposits of Aβ, and inhibition or delay or behavioral deficits. Activeimmunogens can also be tested for induction of antibodies in the sera.Both passive and active immunogens can be tested for passage ofantibodies across the blood brain barrier into the brain of a transgenicanimal. Antibodies or fragments inducing an antibody can also be testedin non-human primates that naturally or through induction developsymptoms of diseases characterized by tau. Tests on an antibody oractive agent are usually performed in conjunction with a control inwhich a parallel experiment is conduct except that the antibody oractive agent is absent (e.g., replaced by vehicle). Reduction, delay orinhibition of signs or symptoms disease attributable to an antibody oractive agent under test can then be assessed relative to the control.

VI. Patients Amenable to Treatment

The presence of neurofibrillary tangles has been found in severaldiseases including Alzheimer's disease, Down's syndrome, mild cognitiveimpairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), andprogressive supranuclear palsy (PSP). The present regimes can also beused in treatment or prophylaxis of any of these diseases. Because ofthe widespread association between neurological diseases and conditionsand tau, the present regimes can be used in treatment or prophylaxis ofany subject showing elevated levels of tau or phosphorylated tau (e.g.,in the CSF) compared with a mean value in individuals withoutneurological disease. The present regimes can also be used in treatmentor prophylaxis of neurological disease in individuals having a mutationin tau associated with neurological disease. The present methods areparticularly suitable for treatment or prophylaxis of Alzheimer'sdisease, and especially in patients.

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. Patients at risk of disease include those having a knowngenetic risk of disease. Such individuals include those having relativeswho have experienced this disease, and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of riskinclude mutations in tau, such as those discussed above, as well asmutations in other genes associated with neurological disease. Forexample, the ApoE4 allele in heterozygous and even more so in homozygousform is associated with risk of Alzheimer's disease. Other markers ofrisk of Alzheimer's disease include mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively, mutationsin the presenilin genes, PS1 and PS2, a family history of AD,hypercholesterolemia or atherosclerosis. Individuals presently sufferingfrom Alzheimer's disease can be recognized by PET imaging, fromcharacteristic dementia, as well as the presence of risk factorsdescribed above. In addition, a number of diagnostic tests are availablefor identifying individuals who have AD. These include measurement ofCSF tau or phospho-tau and Aβ42 levels. Elevated tau or phospho-tau anddecreased Aβ42 levels signify the presence of AD. Some mutationsassociated with Parkinson's disease. Ala30Pro or Ala53, or mutations inother genes associated with Parkinson's disease such as leucine-richrepeat kinase, PARKS. Individuals can also be diagnosed with any of theneurological diseases mentioned above by the criteria of the DSM IV TR.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70 years of age. Treatment typicallyentails multiple dosages over a period of time. Treatment can bemonitored by assaying antibody levels over time. If the response falls,a booster dosage is indicated. In the case of potential Down's syndromepatients, treatment can begin antenatally by administering therapeuticagent to the mother or shortly after birth.

I. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above (e.g., SEQ ID NO:7 and SEQ ID NO:11).Optionally, such nucleic acids further encode a signal peptide and canbe expressed with the signal peptide linked to the constant region.Coding sequences of nucleic acids can be operably linked with regulatorysequences to ensure expression of the coding sequences, such as apromoter, enhancer, ribosome binding site, transcription terminationsignal, and the like. The nucleic acids encoding heavy and light chainscan occur in isolated form or can be cloned into one or more vectors.The nucleic acids can be synthesized by, for example, solid statesynthesis or PCR of overlapping oligonucleotides. Nucleic acids encodingheavy and light chains can be joined as one contiguous nucleic acid,e.g., within an expression vector, or can be separate, e.g., each clonedinto its own expression vector.

J. Conjugated Antibodies

Conjugated antibodies that specifically bind to antigens such as tau,are useful in detecting the presence of tau; monitoring and evaluatingthe efficacy of therapeutic agents being used to treat patientsdiagnosed with Alzheimer's disease, Down's syndrome, mild cognitiveimpairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP); inhibiting or reducing aggregationof tau; inhibiting or reducing tau fibril formation; reducing orclearing tau deposits; stabilizing non-toxic conformations of tau; ortreating or effecting prophylaxis of Alzheimer's disease, Down'ssyndrome, mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP) in apatient.

For example, such antibodies can be conjugated with other therapeuticmoieties, other proteins, other antibodies, and/or detectable labels.See WO 03/057838; U.S. Pat. No. 8,455,622. Such therapeutic moieties canbe any agent that can be used to treat, combat, ameliorate, prevent, orimprove an unwanted condition or disease in a patient, such asAlzheimer's disease, Down's syndrome, mild cognitive impairment, primaryage-related tauopathy, postencephalitic parkinsonism, posttraumaticdementia or dementia pugilistica, Pick's disease, type C Niemann-Pickdisease, supranuclear palsy, frontotemporal dementia, frontotemporallobar degeneration, argyrophilic grain disease, globular glialtauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complexof Guam, corticobasal degeneration (CBD), dementia with Lewy bodies,Lewy body variant of Alzheimer disease (LBVAD), or progressivesupranuclear palsy (PSP).

Conjugated therapeutic moieties can include cytotoxic agents, cytostaticagents, neurotrophic agents, neuroprotective agents, radiotherapeuticagents, immunomodulators, or any biologically active agents thatfacilitate or enhance the activity of the antibody. A cytotoxic agentcan be any agent that is toxic to a cell. A cytostatic agent can be anyagent that inhibits cell proliferation. A neurotrophic agent can be anyagent, including chemical or proteinaceous agents, that promotes neuronmaintenance, growth, or differentiation. A neuroprotective agent can beagent, including chemical or proteinaceous agents, that protects neuronsfrom acute insult or degenerative processes. An immunomodulator can beany agent that stimulates or inhibits the development or maintenance ofan immunologic response. A radiotherapeutic agent can be any molecule orcompound that emits radiation. If such therapeutic moieties are coupledto a tau-specific antibody, such as the antibodies described herein, thecoupled therapeutic moieties will have a specific affinity fortau-related disease-affected cells over normal cells. Consequently,administration of the conjugated antibodies directly targets cancercells with minimal damage to surrounding normal, healthy tissue. Thiscan be particularly useful for therapeutic moieties that are too toxicto be administered on their own. In addition, smaller quantities of thetherapeutic moieties can be used.

Some such antibodies can be modified to act as immunotoxins. See, e.g.,U.S. Pat. No. 5,194,594. For example, ricin, a cellular toxin derivedfrom plants, can be coupled to antibodies by using the bifunctionalreagents S-acetylmercaptosuccinic anhydride for the antibody andsuccinimidyl 3-(2-pyridyldithio)propionate for ricin. See Pietersz etal., Cancer Res. 48(16):4469-4476 (1998). The coupling results in lossof B-chain binding activity of ricin, while impairing neither the toxicpotential of the A-chain of ricin nor the activity of the antibody.Similarly, saporin, an inhibitor of ribosomal assembly, can be coupledto antibodies via a disulfide bond between chemically insertedsulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

Some such antibodies can be linked to radioisotopes. Examples ofradioisotopes include, for example, yttrium⁹⁰ (90Y), indium¹¹¹ (111In),¹³¹I, ⁹⁹mTc, radiosilver-111, radiosilver-199, and Bismuth²¹³. Linkageof radioisotopes to antibodies may be performed with conventionalbifunction chelates. For radiosilver-111 and radiosilver-199 linkage,sulfur-based linkers may be used. See Hazra et al., Cell Biophys.24-25:1-7 (1994). Linkage of silver radioisotopes may involve reducingthe immunoglobulin with ascorbic acid. For radioisotopes such as 111Inand 90Y, ibritumomab tiuxetan can be used and will react with suchisotopes to form 111In-ibritumomab tiuxetan and 90Y-ibritumomabtiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48Suppl 1:S91-S95 (2001).

Some such antibodies can be linked to other therapeutic moieties. Suchtherapeutic moieties can be, for example, cytotoxic, cytostatic,neurotrophic, or neuroprotective. For example, antibodies can beconjugated with toxic chemotherapeutic drugs such as maytansine,geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g.,auristatins), or minor groove binding agents such as calicheamicin.Other representative therapeutic moieties include agents known to beuseful for treatment, management, or amelioration of Alzheimer'sdisease, Down's syndrome, mild cognitive impairment, primary age-relatedtauopathy, postencephalitic parkinsonism, posttraumatic dementia ordementia pugilistica, Pick's disease, type C Niemann-Pick disease,supranuclear palsy, frontotemporal dementia, frontotemporal lobardegeneration, argyrophilic grain disease, globular glial tauopathy,amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam,corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy bodyvariant of Alzheimer disease (LBVAD), or progressive supranuclear palsy(PSP).

Antibodies can also be coupled with other proteins. For example,antibodies can be coupled with Fynomers. Fynomers are small bindingproteins (e.g., 7 kDa) derived from the human Fyn SH3 domain They can bestable and soluble, and they can lack cysteine residues and disulfidebonds. Fynomers can be engineered to bind to target molecules with thesame affinity and specificity as antibodies. They are suitable forcreating multi-specific fusion proteins based on antibodies. Forexample, Fynomers can be fused to N-terminal and/or C-terminal ends ofantibodies to create bi- and tri-specific FynomAbs with differentarchitectures. Fynomers can be selected using Fynomer libraries throughscreening technologies using FACS, Biacore, and cell-based assays thatallow efficient selection of Fynomers with optimal properties. Examplesof Fynomers are disclosed in Grabulovski et al., J. Biol. Chem.282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel.20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner etal., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6):1124-1137 (2013);and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

The antibodies disclosed herein can also be coupled or conjugated to oneor more other antibodies (e.g., to form antibody heteroconjugates). Suchother antibodies can bind to different epitopes within tau or can bindto a different target antigen.

Antibodies can also be coupled with a detectable label. Such antibodiescan be used, for example, for diagnosing Alzheimer's disease, Down'ssyndrome, mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP),and/or for assessing efficacy of treatment. Such antibodies areparticularly useful for performing such determinations in subjectshaving or being susceptible to Alzheimer's disease, Down's syndrome,mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP), orin appropriate biological samples obtained from such subjects.Representative detectable labels that may be coupled or linked to anantibody include various enzymes, such as horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;prosthetic groups, such streptavidin/biotin and avidin/biotin;fluorescent materials, such as umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as luminol;bioluminescent materials, such as luciferase, luciferin, and aequorin;radioactive materials, such as radiosilver-111, radiosilver-199,Bismuth²¹³, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,), carbon (¹⁴C), sulfur (⁵S),tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies; nonradioactive paramagnetic metal ions; and molecules thatare radiolabelled or conjugated to specific radioisotopes.

Linkage of radioisotopes to antibodies may be performed withconventional bifunction chelates. For radiosilver-111 andradiosilver-199 linkage, sulfur-based linkers may be used. See Hazra etal., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes mayinvolve reducing the immunoglobulin with ascorbic acid. Forradioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be usedand will react with such isotopes to form 111In-ibritumomab tiuxetan and90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother.Pharmacol., 48 Suppl 1:S91-S95 (2001).

Therapeutic moieties, other proteins, other antibodies, and/ordetectable labels may be coupled or conjugated, directly or indirectlythrough an intermediate (e.g., a linker), to an antibody of theinvention. See e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy,” in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., Immunol.Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavableand non-cleavable linkers. Different linkers that release the coupledtherapeutic moieties, proteins, antibodies, and/or detectable labelsunder acidic or reducing conditions, on exposure to specific proteases,or under other defined conditions can be employed.

VI. Pharmaceutical Compositions and Methods of Use

In prophylactic applications, an antibody or agent for inducing anantibody or a pharmaceutical composition the same is administered to apatient susceptible to, or otherwise at risk of a disease (e.g.,Alzheimer's disease) in regime (dose, frequency and route ofadministration) effective to reduce the risk, lessen the severity, ordelay the onset of at least one sign or symptom of the disease. Inparticular, the regime is preferably effective to inhibit or delay tauor phospho-tau and paired filaments formed from it in the brain, and/orinhibit or delay its toxic effects and/or inhibit/or delay developmentof behavioral deficits. In therapeutic applications, an antibody oragent to induce an antibody is administered to a patient suspected of,or already suffering from a disease (e.g., Alzheimer's disease) in aregime (dose, frequency and route of administration) effective toameliorate or at least inhibit further deterioration of at least onesign or symptom of the disease. In particular, the regime is preferablyeffective to reduce or at least inhibit further increase of levels oftau, phosphor-tau, or paired filaments formed from it, associatedtoxicities and/or behavioral deficits.

A regime is considered therapeutically or prophylactically effective ifan individual treated patient achieves an outcome more favorable thanthe mean outcome in a control population of comparable patients nottreated by methods of the invention, or if a more favorable outcome isdemonstrated in treated patients versus control patients in a controlledclinical trial (e.g., a phase II, phase II/III or phase III trial) atthe p<0.05 or 0.01 or even 0.001 level.

Effective doses of vary depending on many different factors, such asmeans of administration, target site, physiological state of thepatient, whether the patient is an ApoE carrier, whether the patient ishuman or an animal, other medications administered, and whethertreatment is prophylactic or therapeutic.

Exemplary dosage ranges for antibodies are from about 0.01 to 60 mg/kg,or from about 0.1 to 3 mg/kg or 0.15-2 mg/kg or 0.15-1.5 mg/kg, ofpatient body weight. Antibody can be administered such doses daily, onalternative days, weekly, fortnightly, monthly, quarterly, or accordingto any other schedule determined by empirical analysis. An exemplarytreatment entails administration in multiple dosages over a prolongedperiod, for example, of at least six months. Additional exemplarytreatment regimes entail administration once per every two weeks or oncea month or once every 3 to 6 months.

The amount of an agent for active administration varies from 0.1-500 μgper patient and more usually from 1-100 or 1-10 μg per injection forhuman administration. The timing of injections can vary significantlyfrom once a day, to once a year, to once a decade. A typical regimenconsists of an immunization followed by booster injections at timeintervals, such as 6 week intervals or two months. Another regimenconsists of an immunization followed by booster injections 1, 2 and 12months later. Another regimen entails an injection every two months forlife. Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

Antibodies or agents for inducing antibodies are preferably administeredvia a peripheral route (i.e., one in which an administered or inducedantibody crosses the blood brain barrier to reach an intended site inthe brain. Routes of administration include topical, intravenous, oral,subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal,intranasal, intraocular, or intramuscular. Preferred routes foradministration of antibodies are intravenous and subcutaneous. Preferredroutes for active immunization are subcutaneous and intramuscular. Thistype of injection is most typically performed in the arm or leg muscles.In some methods, agents are injected directly into a particular tissuewhere deposits have accumulated, for example intracranial injection.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The present regimes can be administered in combination with anotheragent effective in treatment or prophylaxis of the disease beingtreated. For example, in the case of Alzheimer's disease, the presentregimes can be combined with immunotherapy against AP (WO/2000/072880),cholinesterase inhibitors or memantine or in the case of Parkinson'sdisease immunotherapy against alpha synuclein WO/2008/103472, Levodopa,dopamine agonists, COMT inhibitors, MAO-B inhibitors, Amantadine, oranticholinergic agents.

Antibodies are administered in an effective regime meaning a dosage,route of administration and frequency of administration that delays theonset, reduces the severity, inhibits further deterioration, and/orameliorates at least one sign or symptom of a disorder being treated. Ifa patient is already suffering from a disorder, the regime can bereferred to as a therapeutically effective regime. If the patient is atelevated risk of the disorder relative to the general population but isnot yet experiencing symptoms, the regime can be referred to as aprophylactically effective regime. In some instances, therapeutic orprophylactic efficacy can be observed in an individual patient relativeto historical controls or past experience in the same patient. In otherinstances, therapeutic or prophylactic efficacy can be demonstrated in apreclinical or clinical trial in a population of treated patientsrelative to a control population of untreated patients.

Exemplary dosages for an antibody are 0.1-60 mg/kg (e.g., 0.5, 3, 10,30, or 60 mg/kg), or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5mg/kg) or 10-4000 mg or 10-1500 mg as a fixed dosage. The dosage dependson the condition of the patient and response to prior treatment, if any,whether the treatment is prophylactic or therapeutic and whether thedisorder is acute or chronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous,intra-arterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Some antibodies can be administered intothe systemic circulation by intravenous or subcutaneous administration.Intravenous administration can be, for example, by infusion over aperiod such as 30-90 min

The frequency of administration depends on the half-life of the antibodyin the circulation, the condition of the patient and the route ofadministration among other factors. The frequency can be daily, weekly,monthly, quarterly, or at irregular intervals in response to changes inthe patient's condition or progression of the disorder being treated. Anexemplary frequency for intravenous administration is between weekly andquarterly over a continuous cause of treatment, although more or lessfrequent dosing is also possible. For subcutaneous administration, anexemplary dosing frequency is daily to monthly, although more or lessfrequent dosing is also possible.

The number of dosages administered depends on whether the disorder isacute or chronic and the response of the disorder to the treatment. Foracute disorders or acute exacerbations of a chronic disorder, between 1and 10 doses are often sufficient. Sometimes a single bolus dose,optionally in divided form, is sufficient for an acute disorder or acuteexacerbation of a chronic disorder. Treatment can be repeated forrecurrence of an acute disorder or acute exacerbation. For chronicdisorders, an antibody can be administered at regular intervals, e.g.,weekly, fortnightly, monthly, quarterly, every six months for at least1, 5 or 10 years, or the life of the patient.

A. Diagnostics and Monitoring Methods

In Vivo Imaging, Diagnostic Methods, and Optimizing Immunotherapy

The invention provides methods of in vivo imaging tau protein deposits(e.g., neurofibrillary tangles and tau inclusions) in a patient. Themethods work by administering a reagent, such as antibody that binds tau(e.g., a mouse, humanized, chimeric or veneered 16G7 antibody), to thepatient and then detecting the agent after it has bound. In somemethods, the antibody binds to an epitope of tau within amino acidresidues 55-78 of SEQ ID NO:3 (corresponding to amino acid residues113-136 of SEQ ID NO:1). In some methods, the antibody binds to anepitope within amino acid residues 60-75 of SEQ ID NO:3 (correspondingto amino acid residues 118-133 of SEQ ID NO:1), or within amino acidresidues 61-70 of SEQ ID NO:3 (corresponding to amino acid residues119-128 of SEQ ID NO:1). A clearing response to the administeredantibodies can be avoided or reduced by using antibody fragments lackinga full-length constant region, such as Fabs. In some methods, the sameantibody can serve as both a treatment and diagnostic reagent.

Diagnostic reagents can be administered by intravenous injection intothe body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labeled, although in some methods, the primary reagentwith affinity for tau is unlabeled and a secondary labeling agent isused to bind to the primary reagent. The choice of label depends on themeans of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT).

The methods of in vivo imaging of tau protein deposits are useful todiagnose or confirm diagnosis of a tauopathy, such as Alzheimer'sdisease, frontotemporal lobar degeneration, progressive supranuclearpalsy and Pick's disease, or susceptibility to such a disease. Forexample, the methods can be used on a patient presenting with symptomsof dementia. If the patient has abnormal neurofibrillary tangles, thenthe patient is likely suffering from Alzheimer's disease. Alternatively,if the patient has abnormal tau inclusions, then depending on thelocation of the inclusions, the patient may be suffering fromfrontotemporal lobar degeneration. The methods can also be used onasymptomatic patients. Presence of abnormal tau protein depositsindicates susceptibility to future symptomatic disease. The methods arealso useful for monitoring disease progression and/or response totreatment in patients who have been previously diagnosed with atau-related disease.

Diagnosis can be performed by comparing the number, size, and/orintensity of labeled loci, to corresponding baseline values. The baseline values can represent the mean levels in a population of undiseasedindividuals. Baseline values can also represent previous levelsdetermined in the same patient. For example, baseline values can bedetermined in a patient before beginning tau immunotherapy treatment,and measured values thereafter compared with the baseline values. Adecrease in values relative to baseline signals a positive response totreatment.

In some patients, diagnosis of a tauopathy may be aided by performing aPET scan. A PET scan can be performed using, for example, a conventionalPET imager and auxiliary equipment. The scan typically includes one ormore regions of the brain known in general to be associated with tauprotein deposits and one or more regions in which few if any depositsare generally present to serve as controls.

The signal detected in a PET scan can be represented as amultidimensional image. The multidimensional image can be in twodimensions representing a cross-section through the brain, in threedimensions, representing the three dimensional brain, or in fourdimensions representing changes in the three dimensional brain overtime. A color scale can be used with different colors indicatingdifferent amounts of label and, inferentially, tau protein depositdetected. The results of the scan can also be presented numerically,with numbers relating to the amount of label detected and consequentlyamount of tau protein deposits. The label present in a region of thebrain known to be associated with deposits for a particular tauopathy(e.g., Alzheimer's disease) can be compared with the label present in aregion known not to be associated with deposits to provide a ratioindicative of the extent of deposits within the former region. For thesame radiolabeled ligand, such ratios provide a comparable measure oftau protein deposits and changes thereof between different patients.

In some methods, a PET scan is performed concurrent with or in the samepatient visit as an MRI or CAT scan. An MRI or CAT scan provides moreanatomical detail of the brain than a PET scan. However, the image froma PET scan can be superimposed on an MRI or CAT scan image moreprecisely indicating the location of PET ligand and inferentially taudeposits relative to anatomical structures in the brain. Some machinescan perform both PET scanning and MRI or CAT scanning without thepatient changing positions between the scans facilitatingsuperimposition of images.

Suitable PET ligands include radiolabeled antibodies of the invention(e.g., a mouse, humanized, chimeric or veneered 16G7 antibody). Theradioisotope used can be, for example, C¹¹, N¹³, O¹⁵, F¹⁸, or I¹²³. Theinterval between administering the PET ligand and performing the scancan depend on the PET ligand and particularly its rate of uptake andclearing into the brain, and the half-life of its radiolabel.

PET scans can also be performed as a prophylactic measure inasymptomatic patients or in patients who have symptoms of mild cognitiveimpairment but have not yet been diagnosed with a tauopathy but are atelevated risk of developing a tauopathy. For asymptomatic patients,scans are particularly useful for individuals considered at elevatedrisk of tauopathy because of a family history, genetic or biochemicalrisk factors, or mature age. Prophylactic scans can commence forexample, at a patient age between 45 and 75 years. In some patients, afirst scan is performed at age 50 years.

Prophylactic scans can be performed at intervals of for example, betweensix months and ten years, preferably between 1-5 years. In somepatients, prophylactic scans are performed annually. If a PET scanperformed as a prophylactic measure indicates abnormally high levels oftau protein deposits, immunotherapy can be commenced and subsequent PETscans performed as in patients diagnosed with a tauopathy. If a PETscanned performed as a prophylactic measure indicates levels of tauprotein deposits within normal levels, further PET scans can performedat intervals of between six months and 10 years, and preferably 1-5years, as before, or in response to appearance of signs and symptoms ofa tauopathy or mild cognitive impairment. By combining prophylacticscans with administration of tau-directed immunotherapy if and when anabove normal level of tau protein deposits is detected, levels of tauprotein deposits can be reduced to, or closer to, normal levels, or atleast inhibited from increasing further, and the patient can remain freeof the tauopathy for a longer period than if not receiving prophylacticscans and tau-directed immunotherapy (e.g., at least 5, 10, 15 or 20years, or for the rest of the patient's life).

Normal levels of tau protein deposits can be determined by the amount ofneurofibrillary tangles or tau inclusions in the brains of arepresentative sample of individuals in the general population who havenot been diagnosed with a particular tauopathy (e.g., Alzheimer'sdisease) and are not considered at elevated risk of developing suchdisease (e.g., a representative sample of disease-free individuals under50 years of age). Alternatively, a normal level can be recognized in anindividual patient if the PET signal according to the present methods ina region of the brain in which tau protein deposits are known to developis not different (within the accuracy of measurement) from the signalfrom a region of the brain in which it is known that such deposits donot normally develop. An elevated level in an individual can berecognized by comparison to the normal levels (e.g., outside mean andvariance of a standard deviation) or simply from an elevated signalbeyond experimental error in a region of the brain associated with tauprotein deposits compared with a region not known to be associated withdeposits. For purposes of comparing the levels of tau protein depositsin an individual and population, the tau protein deposits shouldpreferably be determined in the same region(s) of the brain, theseregions including at least one region in which tau protein depositsassociated with a particular tauopathy (e.g., Alzheimer's disease) areknown to form. A patient having an elevated level of tau proteindeposits is a candidate for commencing immunotherapy.

After commencing immunotherapy, a decrease in the level of tau proteindeposits can be first seen as an indication that the treatment is havingthe desired effect. The observed decrease can be, for example, in therange of 1-100%, 1-50%, or 1-25% of the baseline value. Such effects canbe measured in one or more regions of the brain in which deposits areknown to form or can be measured from an average of such regions. Thetotal effect of treatment can be approximated by adding the percentagereduction relative to baseline to the increase in tau protein depositsthat would otherwise occur in an average untreated patient.

Maintenance of tau protein deposits at an approximately constant levelor even a small increase in tau protein deposits can also be anindication of response to treatment albeit a suboptimal response. Suchresponses can be compared with a time course of levels of tau proteindeposits in patients with a particular tauopathy (e.g., Alzheimer'sdisease) that did not receive treatment, to determine whether theimmunotherapy is having an effect in inhibiting further increases of tauprotein deposits.

Monitoring of changes in tau protein deposits allows adjustment of theimmunotherapy or other treatment regime in response to the treatment.PET monitoring provides an indication of the nature and extent ofresponse to treatment. Then a determination can be made whether toadjust treatment and if desired treatment can be adjusted in response tothe PET monitoring. PET monitoring thus allows for tau-directedimmunotherapy or other treatment regime to be adjusted before otherbiomarkers, MRI or cognitive measures have detectably responded. Asignificant change means that comparison of the value of a parameterafter treatment relative to basement provides some evidence thattreatment has or has not resulted in a beneficial effect. In someinstances, a change of values of a parameter in a patient itselfprovides evidence that treatment has or has not resulted in a beneficialeffect. In other instances, the change of values, if any, in a patient,is compared with the change of values, if any, in a representativecontrol population of patients not undergoing immunotherapy. Adifference in response in a particular patient from the normal responsein the control patient (e.g., mean plus variance of a standarddeviation) can also provide evidence that an immunotherapy regime is oris not achieving a beneficial effect in a patient.

In some patients, monitoring indicates a detectable decline in tauprotein deposits but that the level of tau protein deposits remainsabove normal. In such patients, if there are no unacceptable sideeffects, the treatment regime can be continued as is or even increasedin frequency of administration and/or dose if not already at the maximumrecommended dose.

If the monitoring indicates levels of tau protein deposits in a patienthave already been reduced to normal, or near-normal, levels of tauprotein deposits, the immunotherapy regime can be adjusted from one ofinduction (i.e., that reduces the level of tau protein deposits) to oneof maintenance (i.e., that maintains tau protein deposits at anapproximately constant level). Such a regime can be affected by reducingthe dose and or frequency of administering immunotherapy.

In other patients, monitoring can indicate that immunotherapy is havingsome beneficial effect but a suboptimal effect. An optimal effect can bedefined as a percentage reduction in the level of tau protein depositswithin the top half or quartile of the change in tau protein deposits(measured or calculated over the whole brain or representative region(s)thereof in which tau protein deposits are known to form) experienced bya representative sample of tauopathy patients undergoing immunotherapyat a given time point after commencing therapy. A patient experiencing asmaller decline or a patient whose tau protein deposits remains constantor even increases, but to a lesser extent than expected in the absenceof immunotherapy (e.g., as inferred from a control group of patients notadministered immunotherapy) can be classified as experiencing a positivebut suboptimal response. Such patients can optionally be subject to anadjustment of regime in which the dose and or frequency ofadministration of an agent is increased.

In some patients, tau protein deposits may increase in similar orgreater fashion to tau deposits in patients not receiving immunotherapy.If such increases persist over a period of time, such as 18 months or 2years, even after any increase in the frequency or dose of agents,immunotherapy can if desired be discontinued in favor of othertreatments.

The foregoing description of diagnosing, monitoring, and adjustingtreatment for tauopathies has been largely focused on using PET scans.However, any other technique for visualizing and/or measuring tauprotein deposits that is amenable to the use of tau antibodies of theinvention (e.g., a mouse, humanized, chimeric or veneered 16G7 antibody)can be used in place of PET scans to perform such methods.

Also provided are methods of detecting an immune response against tau ina patient suffering from or susceptible to diseases associated with tau.The methods can be used to monitor a course of therapeutic andprophylactic treatment with the agents provided herein. The antibodyprofile following passive immunization typically shows an immediate peakin antibody concentration followed by an exponential decay. Without afurther dose, the decay approaches pretreatment levels within a periodof days to months depending on the half-life of the antibodyadministered. For example, the half-life of some human antibodies is ofthe order of 20 days.

In some methods, a baseline measurement of antibody to tau in thesubject is made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dose of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other subjects. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one or, preferably, two standarddeviations of the reference value in a population of subjects benefitingfrom treatment) administration of an additional dose of antibody isindicated.

Also provided are methods of detecting tau in a subject, for example, bymeasuring tau in a sample from a subject or by in vivo imaging of tau ina subject. Such methods are useful to diagnose or confirm diagnosis ofdiseases associated with tau, or susceptibility thereto. The methods canalso be used on asymptomatic subjects. The presence of tau indicatessusceptibility to future symptomatic disease. The methods are alsouseful for monitoring disease progression and/or response to treatmentin subjects who have been previously diagnosed with Alzheimer's disease,Down's syndrome, mild cognitive impairment, primary age-relatedtauopathy, postencephalitic parkinsonism, posttraumatic dementia ordementia pugilistica, Pick's disease, type C Niemann-Pick disease,supranuclear palsy, frontotemporal dementia, frontotemporal lobardegeneration, argyrophilic grain disease, globular glial tauopathy,amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam,corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy bodyvariant of Alzheimer disease (LBVAD), or progressive supranuclear palsy(PSP).

Biological samples obtained from a subject having, suspected of having,or at risk of having Alzheimer's disease, Down's syndrome, mildcognitive impairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP) can be contacted with theantibodies disclosed herein to assess the presence of tau. For example,levels of tau in such subjects may be compared to those present inhealthy subjects. Alternatively, levels of tau in such subjectsreceiving treatment for the disease may be compared to those of subjectswho have not been treated for Alzheimer's disease, Down's syndrome, mildcognitive impairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP). Some such tests involve a biopsyof tissue obtained from such subjects. ELISA assays may also be usefulmethods, for example, for assessing tau in fluid samples.

IX. Kits

The invention further provides kits (e.g., containers) comprising anantibody disclosed herein and related materials, such as instructionsfor use (e.g., package insert). The instructions for use may contain,for example, instructions for administration of the antibody andoptionally one or more additional agents. The containers of antibody maybe unit doses, bulk packages (e.g., multi-dose packages), or sub-unitdoses.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products.

Kits can also include a second container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It can also include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

X. Other Applications

The antibodies can be used for detecting tau or fragments thereof, inthe context of clinical diagnosis or treatment or in research. Forexample, the antibodies can be used to detect the presence of tau in abiological sample as an indication that the biological sample comprisestau deposits. Binding of the antibodies to the biological sample can becompared to binding of the antibodies to a control sample. The controlsample and the biological sample can comprise cells of the same tissueorigin. Control samples and biological samples can be obtained from thesame individual or different individuals and on the same occasion or ondifferent occasions. If desired, multiple biological samples andmultiple control samples are evaluated on multiple occasions to protectagainst random variation independent of the differences between thesamples. A direct comparison can then be made between the biologicalsample(s) and the control sample(s) to determine whether antibodybinding (i.e., the presence of tau) to the biological sample(s) isincreased, decreased, or the same relative to antibody binding to thecontrol sample(s). Increased binding of the antibody to the biologicalsample(s) relative to the control sample(s) indicates the presence oftau in the biological sample(s). In some instances, the increasedantibody binding is statistically significant. Optionally, antibodybinding to the biological sample is at least 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, or 100-fold higher than antibodybinding to the control sample.

In addition, the antibodies can be used to detect the presence of thetau in a biological sample to monitor and evaluate the efficacy of atherapeutic agent being used to treat a patient diagnosed withAlzheimer's disease, Down's syndrome, mild cognitive impairment, primaryage-related tauopathy, postencephalitic parkinsonism, posttraumaticdementia or dementia pugilistica, Pick's disease, type C Niemann-Pickdisease, supranuclear palsy, frontotemporal dementia, frontotemporallobar degeneration, argyrophilic grain disease, globular glialtauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complexof Guam, corticobasal degeneration (CBD), dementia with Lewy bodies,Lewy body variant of Alzheimer disease (LBVAD), or progressivesupranuclear palsy (PSP). A biological sample from a patient diagnosedwith Alzheimer's disease, Down's syndrome, mild cognitive impairment,primary age-related tauopathy, postencephalitic parkinsonism,posttraumatic dementia or dementia pugilistica, Pick's disease, type CNiemann-Pick disease, supranuclear palsy, frontotemporal dementia,frontotemporal lobar degeneration, argyrophilic grain disease, globularglial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementiacomplex of Guam, corticobasal degeneration (CBD), dementia with Lewybodies, Lewy body variant of Alzheimer disease (LBVAD), or progressivesupranuclear palsy (PSP) is evaluated to establish a baseline for thebinding of the antibodies to the sample (i.e., a baseline for thepresence of the tau in the sample) before commencing therapy with thetherapeutic agent. In some instances, multiple biological samples fromthe patient are evaluated on multiple occasions to establish both abaseline and measure of random variation independent of treatment. Atherapeutic agent is then administered in a regime. The regime mayinclude multiple administrations of the agent over a period of time.Optionally, binding of the antibodies (i.e., presence of tau) isevaluated on multiple occasions in multiple biological samples from thepatient, both to establish a measure of random variation and to show atrend in response to immunotherapy. The various assessments of antibodybinding to the biological samples are then compared. If only twoassessments are made, a direct comparison can be made between the twoassessments to determine whether antibody binding (i.e., presence oftau) has increased, decreased, or remained the same between the twoassessments. If more than two measurements are made, the measurementscan be analyzed as a time course starting before treatment with thetherapeutic agent and proceeding through the course of therapy. Inpatients for whom antibody binding to biological samples has decreased(i.e., the presence of tau), it can be concluded that the therapeuticagent was effective in treating the Alzheimer's disease, Down'ssyndrome, mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's EFORMAT type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP) inthe patient. The decrease in antibody binding can be statisticallysignificant. Optionally, binding decreases by at least 1%, 2%, 3%, 4%,5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.Assessment of antibody binding can be made in conjunction with assessingother signs and symptoms of Alzheimer's disease, Down's syndrome, mildcognitive impairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP).

The antibodies can also be used as research reagents for laboratoryresearch in detecting tau, or fragments thereof. In such uses,antibodies can be labeled with fluorescent molecules, spin-labeledmolecules, enzymes, or radioisotopes, and can be provided in the form ofkit with all the necessary reagents to perform the detection assay. Theantibodies can also be used to purify tau, or binding partners of tau,e.g., by affinity chromatography.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example 1. Identification of Tau Monoclonal Antibodies

Monoclonal antibody 16G7 was prepared according to a modification of themethod of Kohler and Milstein (G. Kohler and C. Milstein (1975) Nature256:495-497). Human tau containing all 4 microtubule binding repeats,and lacking the N-terminal splice variants (4R0N tau, 383 amino acids)was used in all injections and screening assays. Tau was purified fromSF9 cells infected with a tau-containing baculovirus construct (J. Knopset al. (1991) J Cell Biol 115(5):725-33). Six week old ALI mice wereinjected with 100 μg of purified tau at two week intervals. Tau wasemulsified in complete Freund's adjuvant for the first immunization andin incomplete Freund's adjuvant for all subsequent immunizations. Serumsamples were taken three days after the third injection to assess thetiter of these animals. The highest titer mouse was injectedintravenously with 100 μg of tau in 500 μL of PBS two weeks afterreceiving its third injection. The myeloma fusion occurred three dayslater using SP2/0 as the fusion partner. Supernatants from wellscontaining hybridoma cells were screened for their ability toprecipitate ¹²⁵I-labeled tau. Tau was radio-iodinated using immobilizedglucose oxidase and lactoperoxidase according to the manufacturer'sinstructions (Bio-Rad). Briefly, 10 μg of purified recombinant tau wasradiolabeled with 1 mCi of Na¹²⁵I to a specific activity of 20 μCi/μg,protein. 16G7 was identified as a high-affinity monoclonal antibodyspecific to tau and was cloned by limiting dilution. The isotypes of16G7 was determined to be gamma 1 kappa.

Example 2. Epitope Mapping of Antibody 16G7

A range of overlapping biotinylated peptides spanning the entire 383aa4RON human tau protein were used for mapping the murine 16G7 antibody.Additional peptides were used to model potential post-translationalmodifications of the C- and N-terminal ends of the protein.

Biotinylated peptides were bound to separate wells of astreptavidin-coated ELISA plate. The plate was blocked and treated withmurine 16G7, followed by incubation with a horseradishperoxidase-conjugated anti-mouse antibody. After thorough washing, OPDwas applied to the plate and allowed to develop. The plate was read at450 nm absorbance. Background subtraction was performed with absorbancevalues from wells containing no primary antibody, and a threshold forpositive binding was set to 0.2 absorbance units.

Positive binding was detected for the peptide spanning amino acidresidues 55-69 of SEQ ID NO:3, with lesser binding detected towards thepeptide spanning amino acid residues 64-78 of SEQ ID NO:3 (FIG. 1).Using the numbering of the full-length 4R2N human tau protein (441 aminoacids) (SEQ ID NO:1) these peptides correspond to amino acid residues113-127 and 122-136, respectively, of SEQ ID NO:1.

Example 3. Design of Humanized 16G7 Antibodies

The starting point or donor antibody for humanization was the mouseantibody 16G7. The heavy chain variable amino acid sequence of maturem16G7 is provided as SEQ ID NO:7. The light chain variable amino acidsequence of mature m16G7 is provided as SEQ ID NO:11. The heavy chainKabat/Chothia Composite CDR1, CDR2, and CDR3 amino acid sequences areprovided as SEQ ID NOs:8-10, respectively. The light chain Kabat CDR1,CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs12-14respectively. Kabat numbering is used throughout.

The variable kappa (Vk) of 16G7 belongs to mouse Kabat subgroup 1 whichcorresponds to human Kabat subgroup 4 and the variable heavy (Vh) tomouse Kabat subgroup 2b which corresponds to human Kabat subgroup 1[Kabat E. A., et al., (1991), Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242]. 17 residueChothia CDR-L1 belongs to canonical class 3, 7 residue Chothia CDR-L2 toclass 1, 9 residue Chothia CDR-L3 to class 1 in Vk [Martin A. C, andThornton J. M. (1996) J. Mol. Biol. 263:800-15.]. 10 residue ChothiaCDR-H1 belongs to class 1, 17 residue Chothia CDR-H2 to class 2 [Martin& Thornton, 1996]. CDR-H3 has no canonical classes, but the 9 residueloop probably has a kinked base according to the rules of Shirai et al[Shirai H, et al., (1999) FEBS Lett. 455:188-97.]. A search was madeover the protein sequences in the PDB database [Deshpande N, et al.,(2005) Nucleic Acids Res. 33: D233-7.] to find structures which wouldprovide a rough structural model of 16G7. The structure of the anti0-antigen of Francisella tularenis FAB (pdb code 3UJT) [Rynkiewicz, M.J., et al., (2012) Biochemistry 51: 5684-5694] with a resolution of 2.1Awas used to build up a Fv model of 16G7. It retained the same canonicalstructure for the loops as 16G7. For VLv1, VLv2, and VLv3, human VLacceptors AAB24404.1 and AEK69364.1 from a search of the non-redundantprotein sequence database from NCBI were also applied (following theprevious INN humanization rule). For VHv1, VHv2, VHv3, VHv4, VHv5V andVHv5VR, acceptors AAA18265.1 and ADW08092.1 from a search of thenon-redundant protein sequence database from NCBI were also applied(following the previous INN humanization rule).

Following 2015 INN antibody humanization rule [Jones, T. D., et al,(2016), The INNs and outs of antibody nonproprietary names. mAbs(http://dx.doi.org/10.1080/19420862.2015.1114320)], a search of IMGTdatabase allowed selection of suitable human germline frameworks intowhich to graft the murine CDRs. For Vk, a human kappa light chain withIMGT#IGKV4-1*01 was chosen. This has the same canonical classes forCDR-L1, CDR-L2 and L3, and belongs to human kappa subgroup 1. For Vh,human heavy chain with IMGT# IGHV1-2*02 was chosen, which belongs tohuman heavy chain subgroup 1. It shares the canonical form of 16G7CDR-H1 and H2. VHv2a, VHv2b, VHv2aB7, VHv2bH7, VHv6a, VHv6b and VHv7were designed using IMGT# IGHV1-2*02 as an acceptor.

Heavy and light chain variant sequences resulting from antibodyhumanization process were further aligned to human germ line sequencesusing IMGT Domain GapAlign tool to assess the humanness of the heavy andlight chain as outlined by WHO INN committee guidelines. (WHO-INN:International nonproprietary names (INN) for biological andbiotechnological substances (a review) (Internet) 2014. Available from:http://www.who.int/medicines/services/inn/BioRev2014.pdf) Residues werechanged to align with corresponding human germ line sequence, wherepossible, to enhance humanness.

13 humanized heavy chain variable region variants and 3 humanized lightchain variable region variants were constructed containing differentpermutations of substitutions (hu16G7VHv1, hu16G7VHv2, hu16G7VHv2a,16G7VHv2aB7, hu16G7VHv2b, hu16G7VHv2bH7, hu16G7VHv3, hu16G7VHv4,hu16G7VHv5V, hu16G7VHv5VR, hu16G7VHv6a, hu16G7VHv6b, and hu16G7VHv7,(SEQ ID NOs: 15-27, respectively) and hu16G7VLv1. hu16G7VLv2, andhu16G7VLv3 (SEQ ID NOS:28-30, respectively) (Tables 4 and 3). Theexemplary humanized Vk and Vh designs, with backmutations and othermutations based on selected human frameworks, are shown in Tables 3 and4, respectively. The gray-shaded areas in Tables 3 and 4 indicate theCDRs as defined by Kabat/Chothia Composite. SEQ ID NOs: 15-27 and SEQ IDNOs: 28-30 contain backmutations and other mutations as shown in Table5. The amino acids at positions H1, H5, H7, H9, H10, H11, H12, H13, H20,H31, H37, H38, H40, H48, H60, H64, H66, H67, H69, H71, H73, H75, H80,H82s, H82b, H83, and H85 in hu16G7VHv1, hu16G7VHv2), hu16G7VHv2a,16G7VHv2aB7, hu16G7VHv2b, hu16G7VHv2bH7, hu16G7VHv3, hu16G7VHv4,hu16G7VHv5V, hu16G7VHv5VR, hu16G7VHv6a, hu16G7VHv6b, and hu16G7VHv7 arelisted in Table 6. The amino acids at positions in L4, L9, L15, L18,L19, L21, L22, L43, and L48 hu16G7VLv1. hu16G7VLv2, and hu16G7VLv3 atare listed in Table 7. The percentage humanness for humanized VH chainshu16G7VHv1, hu16G7VHv2, hu16G7VHv2a, 16G7VHv2aB7, hu16G7VHv2b,hu16G7VHv2bH7, hu16G7VHv3, hu16G7VHv4, hu16G7VHv5V, hu16G7VHv5VR,hu16G7VHv6a, hu16G7VHv6b, and hu16G7VHv7, (SEQ ID NOs: 15-27,respectively) and humanized VL chains hu16G7VLv1. hu16G7VLv2, andhu16G7VLv3 (SEQ ID NOS:28-30, respectively) is shown in Table 8.

TABLE 5 V_(H), V_(L) Backmutations and Other Mutations for Humanized16G7 Table 5 Changes from Acceptor Framework and CDR Residues (based onKabat/Chothia V_(H) or V_(L) Variant V_(H) or V_(L) Exon AcceptorSequence Composite CDRs) hu16G7VHv1 NCBI accession code IMGT# IGHV1-2*02H1, H5, H7, H9, H10, H11, H12, (SEQ ID NO: 15) (SEQ (ID NO: 33) H13,H20, H37, H38, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82A, H82B,H83, H85 hu16G7VHv2 NCBI accession code IMGT# IGHV1-2*02 H1, H5, H11,H12, H20, H48, (SEQ ID NO: 16) (SEQ (ID NO: 33) H69, H71, H75, H82A,H82B, H83, H85 hu16G7VHv2a NCBI accession code IMGT# IGHV1-2*02 H1, H12,H40, H48, H66, H67, (SEQ ID NO: 17) (SEQ (ID NO: 33) H69, H71, H73, H8316G7VHv2aB7 NCBI accession code IMGT# IGHV1-2*02 H1, H12, H31, H40, H48,H60, (SEQ ID NO: 18) (SEQ (ID NO: 33) H66, H67, H69, H71, H73, H83hu16G7VHv2b NCBI accession code IMGT# IGHV1-2*02 H1, H12, H40, H48, H69,H73, (SEQ ID NO: 19) (SEQ (ID NO: 33) H83 hu16G7VHv2bH7 NCBI accessioncode IMGT# IGHV1-2*02 H1, H12, H31, H40, H48, H60, (SEQ ID NO: 20) (SEQ(ID NO: 33) H64, H69, H73, H83 hu16G7VHv3 NCBI accession code IMGT#IGHV1-2*02 H1, H5, H7, H9, H10, H11, H12, (SEQ ID NO: 21) (SEQ (ID NO:33) H13, H20, H48, H69, H71, H75, H82A, H82B, H83, H85 hu16G7VHv4 NCBIaccession code IMGT# IGHV1-2*02 H1, H5, H11, H12, H20, H48, (SEQ ID NO:22) (SEQ (ID NO: 33) H66, H67, H69, H71, H73, H75, H82A, H82B, H83, H85hu16G7VHv5V NCBI accession code IMGT# IGHV1-2*02 H1, H5, H7, H9, H10,H11, H12, (SEQ ID NO: 23) (SEQ (ID NO: 33) H13, H20, H38, H40, H48, H66,H67, H69, H71, H73, H75, H80, H82A, H82B, H83, H85 hu16G7VHv5VR NCBIaccession code IMGT# IGHV1-2*02 H1, H5, H7, H9, H10, H11, H12, (SEQ IDNO: 24) (SEQ (ID NO: 33) H13, H20, H40, H48, H66, H67, H69, H71, H73,H75, H80, H82A, H82B, H83, H85 hu16G7VHv6a NCBI accession code IMGT#IGHV1-2*02 H7, H71, H73 (SEQ ID NO: 25) (SEQ (ID NO: 33) hu16G7VHv6bNCBI accession code IMGT# IGHV1-2*02 H71 (SEQ ID NO: 26) (SEQ (ID NO:33) hu16G7VHv7 NCBI accession code IMGT# IGHV1-2*02 H1, H7, H71, H73(SEQ ID NO: 27) (SEQ (ID NO: 33) Hu16G7VLv1 NCBI accession code IMGT#IGKV4-1*01 L4, L9, L15, L18, L19, L21, L22, (SEQ ID NO: 28) (SEQ ID NO:36) L43, L48 Hu16G7VLv2 NCBI accession code IMGT# IGKV4-1*01 L4, L9,L15, L22, L43 (SEQ ID NO: 29) (SEQ ID NO: 36)) Hu16G7VLv3 NCBI accessioncode IMGT# IGKV4-1*01 L4, L9, L15, L18, L19, L21, L22, (SEQ ID NO: 30)(SEQ ID NO: 36) L43

TABLE 6 Kabat Numbering of Framework (or CDR) Residues (based onKabat/Chothia Composite CDRs) for Backmutations and Other Mutations inHeavy Chains of Humanized 16G7 Antibodies (Heavy Mouse Residue Chain)16G7 hu16G7VHv1 hu16G7VHv2 hu16G7VHv2a 16G7VHv2aB7 hu16G7VHv2bhu16G7VHv2bH7 hu16G7VHv3 H1  Q Q E E E E E E E H5  V Q Q Q V V V V Q H7 S P P S S S S S P H9  A S S A A A A A S H10  E V V E E E E E V H11  V LL L V V V V L H12  K V V V V V V V V H13  K R R K K K K K R H20  V L L LV V V V L H31  G S S S S G S G S H37  V A A V V V V V V H38  R K K R R RR R R H40  A R R A R R R R A H48  M I I I I I I I I H60  A N N N N A N AN H64  Q K K K K K K Q K H66  R K K R K K R R R H67  V A A V A A V V VH69  M L L L L L L L L H71  R V V V V V R R V H73  T I I T I I I I TH75  I S S A I I I I A H80  M V V M M M M M M H82A S T T T S S S S TH82B R S S S R R R R S H83  R T T T T T T T T H85  D E E E D D D D EResidue hu16G7VHv4 hu16G7VHv5V hu16G7VHv5VR hu16G7VHv6a hu16G7VHv6bhu16G7VHv7 H1  E E E Q Q E H5  Q Q Q V V V H7  S P P p S P H9  A S S A AA H10  E V V E E E H11  L L L V V V H12  V V V K K K H13  K R R K K KH20  L L L V V V H31  S S S S S S H37  V V V V V V H38  R K R R R R H40 A R R A A A H48  I I I M M M H60  N N N N N N H64  K K K K K K H66  K KK R R R H67  A A A V V V H69  L L L M M M H71  V V V V V V H73  I I I IT I H75  S S S I I I H80  M V V M M M H82A T T T S S S H82B S S S R R RH83  T T T R R R H85  E E E D D D

TABLE 7 Kabat Numbering of Framework Residues (based on Kabat/ChothiaComposite CDRs) for Backmutations and Other Mutations in Light Chains ofHumanized 16G7 Antibodies Table 7 IMGT#IGKV4-1*01 Mouse Residue (LightChain) 16G7 Hu16G7VLv1 Hu16G7VLv2 Hu16G7VLv3 L4 M L L L L L9 D S S S SL15 L V V V V L18 R K K R K L19 A V V A V L21 I M M I M L22 N S S S SL43 P S S S S L48 I M M I I

TABLE 8 Percentage Humanness of Heavy and Light Chains of Humanized 16G7Antibodies V_(H) or V_(L) Variant % Humanness hu16G7VHv1 (SEQ ID NO: 15)64.30% hu16G7VHv2 (SEQ ID NO: 16) 76.50% hu16G7VHv2a (SEQ ID NO: 17)79.60% 16G7VHv2aB7 (SEQ ID NO: 18) 81.60% hu16G7VHv2b (SEQ ID NO: 19)82.70% hu16G7VHv2bH7 (SEQ ID NO: 20) 85.70% hu16G7VHv3 (SEQ ID NO: 21)72.40% hu16G7VHv4 (SEQ ID NO: 22) 73.50% hu16G7VHv5V (SEQ ID NO: 23)66.30% hu16G7VHv5VR (SEQ ID NO: 24) 67.30% hu16G7VHv6a (SEQ ID NO: 25)86.70% hu16G7VHv6b (SEQ ID NO: 26) 88.80% hu16G7VHv7 (SEQ ID NO: 27)85.70% hu16G7VLv1 (SEQ ID NO: 28) 82.20% hu16G7VLv2 (SEQ ID NO: 29)86.10% hu16G7VLv3 (SEQ ID NO: 30) 83.20%

Positions at which canonical, vernier, or interface residues differbetween mouse and human acceptor sequences are candidates forsubstitution. Examples of canonical/CDR interacting residues includeKabat residues H24, H26, H27, H29, H34, H52a, H55, H70, H71, H74, H94,L2, L4, L25, L27b, L33, L48, L64, L71, L90, and L95 in Table 4. Examplesof interface/packing (VH+VL) residues include Kabat residues H35, H37,H39, H45, H47, H91, H93, H95, H100a, H103, L34, L36, L38, L44, L46, L87,L89, L91, L96, L98, in Table 3.

The rationales for selection of the positions indicated in Table 3 inthe light chain variable region as candidates for substitution are asfollows.

M4L is a mutation of a residue that contacts LCDR1 and LCDR3 P43S is amutation of a residue that contacts two interface residues in (Y91 andW103) in VH. I48M is a mutation of an interface residue. D9S, L15V,R18K, A19V, I21M, and N22S are frequency based back-mutations orfrequency/germ-line aligning mutations.

The rationales for selection of the positions indicated in Table 4 inthe heavy chain variable region as candidates for substitution are asfollows.

Q1E is a stability enhancing mutation to mitigate pyroglutamateformation potential. (Liu, 2011, supra).

S7P is a mutation to proline to improve folding. R71V is a mutation of aresidue that may contact HCDR2 via hydrogen bond. T73I is a mutation ofa residue that may contact HCDR1 and HCDR2.

V5Q, E10V, V11L, K12V, K13R, V20L, S31G, V37A, R38K, A40R, M48I, N60A,K64Q, R66K, V67A. M69L, I75S, I75A, M80V, S82a-T, R83b-S, R83T, and D85Eare frequency based back-mutations or germ-line aligning mutations.

Humanized sequences are generated using a two-stage PCR protocol thatallows introduction of multiple mutations, deletions, and insertionsusing QuikChange site-directed mutagenesis [Wang, W. and Malcolm, B. A.(1999) BioTechniques 26:680-682).

The designs based on these human frameworks were:

VARIABLE KAPPA hu16G7VLv1 (SEQ ID NO: 28):DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLMYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ FYDYPWTFGGGTKLEIKRhu16G7VLv2 (SEQ ID NO: 29):DIVLTQSPSSLAVSVGERATISCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ FYDYPWTFGGGTKLEIKRhu16G7VLv3 (SEQ ID NO: 30):DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYDYPWTFGGGTKLEIKRVARIABLE HEAVY hu16G7VHv1 (SEQ ID NO: 15):EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv2 (SEQ ID NO:16):EVQLQQSGAELVKPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTNYNEKFKGRVTLTVDTSASTAYMELTSLTSEDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv2a (SEQ ID NO: 17):EVQLVQSGAEVVKPGASVKVSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISISTAYMELSRLTSDDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VH2aB7 (SEQ ID NO: 18)EVQLVQSGAEVVKPGASVKVSCKASGYTFTGSWIHWVRQRPGQGLEWIGEIYPNSGNTNYAEKFKGKATLTVDISISTAYMELSRLTSDDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv2b (SEQ ID NO: 19):EVQLVQSGAEVVKPGASVKVSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTNYNEKFKGRVTLTRDISISTAYMELSRLTSDDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VH2bH7 (SEQ ID NO: 20)EVQLVQSGAEVVKPGASVKVSCKASGYTFTGSWIHWVRQRPGQGLEWIGEIYPNSGNTNYAEKFQGRVTLTRDISISTAYMELSRLTSDDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv3(SEQ ID NO: 21):EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTNYNEKFKGRVTLTVDTSASTAYMELTSLTSEDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv4 (SEQ ID NO: 22):EVQLQQSGAELVKPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYMELTSLTSEDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv5V (SEQ ID NO: 23):EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVKQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv5VR (SEQ ID NO: 24):EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv6a (SEQ ID NO: 25):QVQLVQPGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGNTNYNEKFKGRVTMTVDISISTAYMELSRLRSDDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv6b (SEQ ID NO: 26):QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGNTNYNEKFKGRVTMTVDTSISTAYMELSRLRSDDTAVYYC ARLGWLIPLDYWGQGTTVTVSShu16G7VHv7 (SEQ ID NO: 27):EVQLVQPGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGNTNYNEKFKGRVTMTVDISISTAYMELSRLRSDDTAVYYC ARLGWLIPLDYWGQGTTVTVSS

Example 4. 16G7 Immunocaptures Tau from Human Alzheimer's Disease Tissue

Methods:

FIG. 4A: Temporal cortical tissue from an Alzheimer's disease brain wasused to extract protein fractions of varying solubility. Frozen tissuewas homogenized in 5 volumes of reassembly (RAB) buffer (100 mM MES, 1mM EGTA, 0.5 mM MgSO₄, 750 mM NaCl, pH 6.8), and centrifuged to generatethe RAB-soluble fraction. The pellet was then homogenized in 5 volumesof radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris, 150 mMNaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.5) andcentrifuged to generate the RIPA-soluble fraction. This pellet washomogenized in 5 volumes of sarkosyl buffer (25 mM Tris, 1 mM EGTA, 1%sarkosyl, 10% sucrose, 1 mM DTT, 500 mM NaCl, pH 7.5), and centrifugedto generated the sarkosyl-soluble fraction. This finalsarkosyl-insoluble pellet was homogenized in 2% SDS. Samples wereprepared in reducing/denaturing sample buffer, and resolved by SDS-PAGE.Western blotting was performed using murine 16G7.

FIG. 4B: RAB-soluble protein fractions were prepared to 1 mg/ml. Foreach immunoprecipitation, 300 μg of sample was used. 10 μg of theindicated antibody (either an isotype control, 16G7, or anti-tauantibody 16B5) was added to the RAB sample preparations, and incubatedfor 2 hr. Protein G magnetic beads were then added to the mixtures, andincubated for a further hour to capture antibody/antigen complexes.Samples were thoroughly washed with 1×PBS, and beads were boiled inreducing/denaturing sample buffer to release captured proteins.Resulting samples were resolved by SDS-PAGE and Western blotting wasperformed using a polyclonal anti-tau antibody (Dako, #A0024).

Results:

FIG. 4A: 16G7 recognizes soluble and sarkosyl-insoluble tau from humanAlzheimer disease tissue. Protein fractions of varying solubility wereresolved by SDS-PAGE and blotted with 16G7. Multiple tau isoforms areseen in soluble and insoluble fractions, and an electrophoretic mobilityshift is detected in insoluble fractions, consistent with a pathologicalform of tau. There is a noted accumulation of tau in the sarkosylinsoluble fraction. Left: Molecular weight markers. Lane designations:1—RAB soluble, 2—RIPA soluble, 3—Sarkosyl Soluble, 4—Sarkosyl Insoluble.

FIG. 4B: 16G7 immunoprecipitates tau from Alzheimer's disease tissue.RAB-soluble fractions were immunoprecipitated with the indicatedantibody, and detected with a polyclonal anti-tau antibody directedtowards a separate region of the tau molecule from the binding sites for16G7 and anti-tau antibody 16B5. 16G7 robustly captures tau from thisfraction. The input (RAB-soluble sample) is shown at right.

Example 5. Reactivity Tow Towards Human Alzheimer's Disease Tissue(Immunohistochemistry)

Frontotemporal cortices were obtained from patients withoutneurodegenerative disease or with Alzheimer's disease, which wasconfirmed upon post-mortem assessment. Immunohistochemistry wasperformed on lightly acetone-fixed, 10 μm slide-mounted cryosections.All staining steps were performed using a Leica BOND Rx autostainer,using Leica consumables. Either murine or human forms of 16G7 wereincubated with tissue sections followed by addition ofspecies-appropriate secondary antibodies conjugated to an HRP polymer.To prevent non-specific binding of endogenous immunoglobulin when usinghumanized antibodies on human tissue, the antibodies were non-covalentlylabeled with a biotin-conjugated anti-human monovalent Fab fragment invitro before incubation on tissue. Tissue labeled with the primaryantibody-biotin Fab fragment complex was further amplified using anavidin-biotin amplification system (Vector Laboratories, Burlingame,Calif.). The staining was visualized with a DAB chromogen, whichproduced a brown deposit. Negative control consisted of performing theentire immunohistochemical procedure on adjacent sections with an IgGisotype control antibody.

Antibodies tested were murine 16G7, chimeric 16G7 (which contained VHand VL from the murine antibody with human constant regions, heavy chainSEQ ID NO:54 and light chain SEQ ID NO:55), and humanized 16G7 variantshu16G7VHv7/hu16G7VLv2 and hu16G7VHv2bH7/hu16G7VLv2.

Staining performed with murine 16G7, chimeric 16G7, and humanized formsof 16G7 (hu16G7VHv7/hu16G7VLv2 and hu16G7VHv2bH7/hu16G7VLv2) werequalitatively compared and assessed for the strength and intensity ofstaining, as well as localization of immuoreactivity. Intensity ofstaining was similar for chimeric and humanized forms of 16G7, anddisplayed similar localization patterns compared with the murine form ofthe antibody. Tau was detected in neurofibrillary tangles, fibrils,neuropil threads, and in degenerating axons. There was also notablesomal staining detected.

Example 6. Disaggregation Activity

Methods:

Aggregation of recombinant tau—Purified recombinant tau with anN-terminal 6×His tag was combined with equimolar amounts oflow-molecular weight heparin in 1×PBS (pH 7.4), and incubated at 37° C.for 96 hr on a nutator. Aggregation of the sample was confirmed bybinding to Thioflavin T.

Incubation with antibodies—antibodies were incubated with aggregated,recombinant tau at the indicated molar ratios incubated at 37° C. for 96hr without rotation or nutation. At the end of the experiment,aggregation was measured by incubating samples with 25 mM Thioflavin T,and measuring emitted fluorescence (450 nm/482 nm excitation/emission).Signals were background subtracted to buffer samples.

Results:

As shown in FIG. 5, 16G7 preferentially disassembles intact tau fibrils.Varying molar ratios of 16G7 (triangles), isotype control (circles) andanti-tau antibody 16B5 (squares) were incubated with amyloid-containingtau fibrils for 96 hours. At the end of this period, the extent ofaggregation was assessed by binding to thioflavin T. 16G7 preferentiallydecreases the Thioflavin T signal present in the sample, compared toboth an isotype control antibody as well as an anti-tau antibody 16B5that binds to a different region of tau.

Example 7. Affinity of Murine 16G7 Antibody to Tau

SPR analysis was performed using a Biacore T200 to determine the bindingkinetics of 16G7 to recombinant human tau. To prepare a sensor surface,anti-mouse antibody (GE Life Sciences) was immobilized on sensor chipCM5 via amine coupling, and 16G7 was captured at a level to ensuremaximum binding of 50 RU. Various concentrations of recombinant tauranging from 10-0.14 nM were passed over the captured ligand at a flowrate of 50 μL/min in running buffer (HBS+0.05% P-20, 1 mg/mL BSA), for180 sec association and 900 sec dissociation. Data weredouble-referenced to both an irrelevant sensor not containing antibodyligand, and 0 nM analyte concentration to account for the dissociationof ligand from the capture moiety. Data was then analyzed using a global1:1 fit. Binding kinetics are shown in FIG. 6.

Example 8. Affinity of Chimeric 16G7 Antibody and Humanized VariantsTowards Tau

SPR analysis was performed using a Biacore T200 to determine the bindingkinetics of 16G7 to recombinant human tau. To prepare a sensor surface,anti-human antibody (GE Life Sciences) was immobilized on sensor chipCM5 via amine coupling, and chimeric or humanized antibodies werecaptured at a level to ensure maximum binding of 50 RU. Variousconcentrations of recombinant tau ranging from 10-0.14 nM were passedover the captured ligands in parallel at a flow rate of 50 μL/min inrunning buffer (HBS+0.05% P-20, 1 mg/mL BSA), for 180 sec associationand 900 sec dissociation. Data were double-referenced to both anirrelevant sensor not containing antibody ligand, and 0 nM analyteconcentration to account for the dissociation of ligand from the capturemoiety. Data was then analyzed using a global 1:1 fit.

Antibodies tested were chimeric 16G7 ((which contained VH and VL fromthe murine antibody with human constant regions, heavy chain SEQ IDNO:54 and light chain SEQ ID NO:55), and humanized variants(hu16G7VHv2a/hu16G7VLv2, hu16G7VHv2b/hu16G7VLv2, andhu16G7VHv7/hu16G7VLv2),

FIG. 7: Affinity of the tested antibodies was determined by SPRanalysis. Varying concentrations of recombinant tau were flowed overimmobilized antibody, and resulting sensorgrams were analyzed using aglobal 1:1 fit. The kinetic affinity, association, and dissociationrates are shown in FIG. 7.

Example 9. Avidity Towards Aggregated Tau

Methods:

Generation of Fab fragments Fab fragments of 16G7 were generated usingthe Fab Micro Preparation kit following manufacturer's directions(Pierce). Removal of liberated Fc and verification of intact finalproduct were monitored by SDS-PAGE, and concentration was determinedusing the bicinchoninic acid assay (Pierce).

Aggregation of recombinant tau Purified recombinant tau with anN-terminal 6×His tag was combined with equimolar amounts oflow-molecular weight heparin in 1×PBS (pH 7.4), and incubated at 37° C.for 96 hr on a nutator. Aggregation of the sample was confirmed bybinding to Thioflavin T.

SPR analysis of Fab fragments and intact antibody Comparative bindingstudies of Fab fragments and intact antibodies were performed with aBiacore T200. To prepare a sensor surface, anti-his antibodies (GE LifeSciences) were immobilized on sensor chip CM3 via amine coupling, andaggregated recombinant tau (prepared above) was passed over a testchannel and acted as the assay ligand. 16G7 (either intact antibody orFab fragments) were passed over the captured aggregated tau at a flowrate of 50 μL/min in running buffer (HBS+0.05% P-20, 1 mg/mL BSA) insingle cycle mode, at concentrations ranging from 10-0.016 nM for 180sec association phases and a final dissociation phase of 900 sec.Association/dissociation traces were double-reference subtracted to bothan irrelevant sensor not containing antibody ligand, and 0 nM analyteconcentration to account for the dissociation of ligand from the capturemoiety.

Results:

Avidity is the measure of multiple affinities of an antibody-antigeninteraction, and more closely represents the functional affinity of anantibody towards an immobilized antigen such as aggregates orneurofibrillary tangles composed of tau. In SPR measurements, thedifference between affinity (single interaction) and avidity (multipleinteractions) is made by comparing the kinetics of binding of a Fabfragment containing a single binding domain, and an intact antibody withtwo binding domains.

In FIG. 8, the binding measurements of 16G7 Fab fragments are comparedwith intact antibody. The association (solid horizontal lines) anddissociation (dashed horizontal lines) phases are diagrammed. Thedifference between single binding-site affinity and dual binding-siteavidity is most apparent in the terminal dissociation phase, starting at1300 sec. There is no apparent dissociation of antibody/antigen complexfor intact antibody, whereas Fab fragments clearly dissociate fromaggregated tau.

Listing of Sequences P10636-8 (SEQ ID NO: 1)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL P10636-7 (SEQ ID NO: 2)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLP10636-6 (4RON human tau) (SEQ ID NO: 3)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL P10636-5 (SEQ ID NO: 4)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLP10636-4 (SEQ ID NO: 5)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL P10636-2 (SEQ ID NO: 6)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLSEQ ID NO: 7; VH protein sequence:QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYVDLTSLTSEDSAVYYCARLGWLIPLDYWGQGTTLIVSSSEQ ID NO: 8; Murine HCDR1: GYTFTSSWIH SEQ ID NO: 9; Murine HCDR2:EIYPNSGNTNYNEKFKG SEQ ID NO: 10; Murine HCDR3: LGWLIPLDYSEQ ID NO: 11; Murine VL protein sequence:DIVLSQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLMYWASTRESGVPDRFTGSGSGTDFTLTISSLKAEDLAVYYCQQFYDYPWTFGGGTKLEIKRSEQ ID NO: 12; Murine LCDR1: KSSQSLLDGNDQKNYLASEQ ID NO: 13; Murine LCDR2: WASTRES SEQ ID NO: 14; Murine LCDR3:QQFYDYPWT SEQ ID NO:15; >hu16G7VHv1EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 16; >hu16G7VHv2EVQLQQSGAELVKPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTNYNEKFKGRVTLTVDTSASTAYMELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 17; >hu16G7VHv2aEVQLVQSGAEVVKPGASVKVSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISISTAYMELSRLTSDDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 18; >hu16G7VHv2aB7EVQLVQSGAEVVKPGASVKVSCKASGYTFTGSWIHWVRQRPGQGLEWIGEIYPNSGNTNYAEKFKGKATLTVDISISTAYMELSRLTSDDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 19; >hu16G7VHv2bEVQLVQSGAEVVKPGASVKVSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTNYNEKFKGRVTLTRDISISTAYMELSRLTSDDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 20; >hu16G7VHv2bH7EVQLVQSGAEVVKPGASVKVSCKASGYTFTGSWIHWVRQRPGQGLEWIGEIYPNSGNTNYAEKFQGRVTLTRDISISTAYMELSRLTSDDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 21; >hu16G7VHv3EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTNYNEKFKGRVTLTVDTSASTAYMELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 22; >hu16G7VHv4EVQLQQSGAELVKPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYMELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 23; >hu16G7VHv5VEVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVKQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 24; >hu16G7VHv5VREVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSSSEQ ID NO: 25; >hu16G7VHv6aQVQLVQPGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGNTNYNEKFKGRVTMTVDISISTAYMELSRLRSDDTAVYYCARLGWLIPLDYWGQGTTVT VSSSEQ ID NO: 26; >hu16G7VHv6bQVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGNTNYNEKFKGRVTMTVDTSISTAYMELSRLRSDDTAVYYCARLGWLIPLDYWGQGTTVT VSSSEQ ID NO: 27; >hu16G7VHv7EVQLVQPGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGNTNYNEKFKGRVTMTVDISISTAYMELSRLRSDDTAVYYCARLGWLIPLDYWGQGTTVT VSSSEQ ID NO: 28; >hu16G7VLv1DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLMYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYDYPWTFGGGTKLEIKRSEQ ID NO: 29; >hu16G7VLv2DIVLTQSPSSLAVSVGERATISCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYDYPWTFGGGTKLEIKRSEQ ID NO: 30; >hu16G7VLv3DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYDYPWTFGGGTKLEIKRSEQ ID NO: 31; VH Human acceptor AAA18265.1QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGRGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSIRTAYVELSRLTSDDTAVYYCASLADDDPEDFWGQGTLV TVSSSEQ ID NO: 32; VH Human acceptor ADW08092.1QVQLVESGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQRLEWMGEINPDNGHTNYNEKFKSRVTITVDKSASTAYMELSSLRSEDTAVYYCAREADYSYGAFDIWGPGTT VTVSSSEQ ID NO: 33; VH human acceptor IMGT# IGHV1-2*02QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCSEQ ID NO: 34; VL human acceptor AAB24404.1DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPPMFGQGTKVEIKRSEQ ID NO: 35; VL Human acceptor AEK69364.1DIVLTQSPDSLAVSLGERATIKCKSSQSVLYGSDSKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTEFTLTISSLQAADVAVYYCQEYYSSLCTFGQGTKLEIKRSEQ ID NO: 36; VL Human acceptor IMGT#IGKV4-1*01DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPSEQ ID NO: 37; Murine VH nucleotide sequence:CAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTCCTGGATACACTGGGCGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTTATCCTAATAGTGGTAATACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACATATCCTCCAGCACAGCCTACGTGGATCTCACCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGATTGGGATGGTTAATACCCCTTGACTACTGGGGCCAAGGCACCACTCTCATAGTCTCCTCA SEQ ID NO: 38; Murine VL nucleotide sequence:GACATTGTGCTGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTAGATGGTAATGATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATGTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTTTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAGTTTTATGACTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTSEQ ID NO: 39; Kabat CDR-H1 SSWIH SEQ ID NO: 40; Chothia CDR-H1 GYTFTSSSEQ ID NO: 41; Chothia CDR-H2 YPNSGN SEQ ID NO: 42; AbM CDR-H2EIYPNSGNTN SEQ ID NO: 43; Contact CDR-L1 KNYLAWYSEQ ID NO: 44; Contact CDR-L2 LLMYWASTRE SEQ ID NO: 45; Contact CDR-L3QQFYDYPW SEQ ID NO: 46; Contact CDR-H1 TSSWIHSEQ ID NO: 47; Contact CDR-H2 WIGEIYPNSGNTNSEQ ID NO: 48; Contact CDR-H3 ARLGWLIPLD SEQ ID NO: 49; Alternate CDR-H1GYTFTGSWIH SEQ ID NO: 50; Alternate CDR-H2 EIYPNSGNTNYAEKFKGSEQ ID: 51; Alternate CDR-H2 EIYPNSGNTNYAEKFQGSEQ ID NO: 52; consensus amino acid sequence among the heavy chainvariable regions of the mouse 16G7 and humanized 16G7 antibodies (labeled “Majority’ in FIG. 2).EVQLXQXGAEXVKPGASVKXSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTNYNEKFKGXXTLTVDISISTAYMELXXLTSXDTAVYYCARLGWLIPLDYWGQGTTVTV SSSEQ ID NO: 53; consensus amino acid sequence among the light chainvariable regions of the mouse 16G7 and humanized 16G7 antibodies(labeled “Majority’ in FIG. 3).DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLXYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYDYPWTFGGGTKLEIKRSEQ ID NO: 54; heavy chain of chimeric 16G7 antibodyQVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIYPNSGNTNYNEKFKGKATLTVDISSSTAYVDLTSLTSEDSAVYYCARLGWLIPLDYWGQGTTLIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 55; light chain of chimeric 16G7 antibodyDIVLSQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLMYWASTRESGVPDRFTGSGSGTDFTLTISSLKAEDLAVYYCQQFYDYPWTFGGGTKLEIKRKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

1-2. (canceled)
 3. An isolated monoclonal antibody, comprising threelight chain CDRs as and three heavy chain CDRs of monoclonal antibody16G7, wherein 16G7 is a mouse antibody characterized by a heavy chainvariable region having an amino acid sequence comprising SEQ ID NO: 7and a light chain variable region having an amino acid sequencecomprising SEQ ID NO:
 11. 4. The antibody of claim 3, wherein the threeheavy chain CDRs are as defined by Kabat/Chothia Composite (SEQ ID NOs:8, 9, and 10) and the three light chain CDRs are as defined byKabat/Chothia Composite (SEQ ID NOs: 12, 13, and 14).
 5. The antibody ofclaim 3 that is 16G7 or a chimeric, veneered, or humanized form thereof.6-7. (canceled)
 8. The antibody of claim 5, wherein each of the variableheavy chain and variable light chain has ≥85% identity to human germlinesequence.
 9. The antibody of claim 5, wherein the three heavy chain CDRsare as defined by Kabat/Chothia Composite (SEQ ID NOs: 8, 9, and 10) andthe three light chain CDRs are as defined by Kabat/Chothia Composite(SEQ ID NOs: 12, 13, and 14); provided that position H31 is occupied byS or G, position H60 is occupied by N or A and position 64 is occupiedby K or Q.
 10. The antibody of claim 9, wherein CDR-H1, CDR-H2 andCDR-H3 have amino acid sequences comprising SEQ ID NO: 49, 50 and 51respectively. 11-12. (canceled)
 13. The antibody of claim 3, wherein theantibody is a humanized antibody. 14-15. (canceled)
 16. The humanizedantibody of claim 443, wherein the CDRs are of a definition selectedfrom the group of Kabat, Chothia, Kabat/Chothia Composite, AbM andContact.
 17. (canceled)
 18. The humanized antibody of claim 16 whereinthe humanized mature heavy chain variable region comprises the threeKabat heavy chain CDRs of 16G7 (SEQ ID NO:39, SEQ ID NO:9, and SEQ IDNO:10) and the humanized mature light chain variable region comprisesthe three Kabat light chain CDRs of 16G7 (SEQ ID NOs: 12-14). 19-21.(canceled)
 22. The humanized antibody of claim 16, comprising ahumanized mature heavy chain variable region having an amino acidsequence at least 90% identical to any one of SEQ ID NO:15-27 and ahumanized mature light chain variable region having an amino acidsequence at least 90% identical to any one of SEQ ID NO: 28-30.
 23. Thehumanized antibody of claim 22, wherein at least one of the followingpositions is occupied by the amino acid as specified: H1 is occupied byE, H5 is occupied by Q, H7 is occupied by P, H9 is occupied by S, H10 isoccupied by V, H11 is occupied L, H12 is occupied by V, H13 is occupiedby R, H20 is occupied by L, H40 is occupied by R, H48 is occupied by I,H66 is occupied by K, H67 is occupied by A, H69 is occupied by L, H71 isoccupied by V, H73 is occupied by I, H75 is occupied by S, H80 isoccupied by V, H82a is occupied by T, H82b is occupied by S, H83 isoccupied by T, and H85 is occupied by E.
 24. The humanized antibody ofclaim 23, provided positions H1, H5, H7, H9, H10, H11, H12, H13, H20,H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83, and H85 inthe VH region are occupied by, E, Q, P, S, V, L, V, R, L, R, I, K, A, L,V, I, S, V, T, S, T, and E, respectively.
 25. (canceled)
 26. Thehumanized antibody of claim 23, provided positions H1, H5, H7, H9, H10,H11, H12, H13, H20, H48, H69, H71, H82a, H82b, H83, and H85 in the VHregion are occupied by E, Q, P, S, V, L, V, R, L, I, L, V, T, S, T, andE, respectively.
 27. (canceled)
 28. The humanized antibody of claim 23,provided positions H1, H5, H11, H12, H20, H48, H69, H71, H82a, H82b,H83, and H85 in the VH region are occupied by E, Q, L, V, L, I, L, V, T,S, T, and E, respectively.
 29. (canceled)
 30. The humanized antibody ofclaim 23, provided positions H1, H12, H31, H40, H48, H60, H69, H73, andH83 in the VH region are occupied by are occupied by E, V, G, R, I, A,L, I, and T, respectively.
 31. (canceled)
 32. The humanized antibody ofclaim 23, provided positions H1, H12, H40, H48, H69, H73, and H83 in theVH region are occupied by are occupied by E, V, R, I, L, I, and T,respectively.
 33. (canceled)
 34. The humanized antibody of claim 23,provided positions H1, H12, H40, H48, H66, H67, H69, H71, H73, and H83in the VH region are occupied by are occupied by E, V, R, I, K, A, L, V,I, and T, respectively.
 35. (canceled)
 36. The humanized antibody ofclaim 23, provided positions H1, H12, H40, H48, H69, H73, and H83 in theVH region are occupied by are occupied by E, V, R, I, L, I, and T,respectively.
 37. (canceled)
 38. The humanized antibody of claim 23,provided positions H7, H71, and H73 in the VH region are occupied by areoccupied by P, V, and I, respectively.
 39. (canceled)
 40. The humanizedantibody of claim 23, provided positions H31 and H60 in the VH regionare occupied by G and A, respectively.
 41. The humanized antibody ofclaim 22 wherein at least one of the following positions is occupied bythe amino acid as specified: H1 is occupied by Q or E, H5 is occupied byV or Q, H7 is occupied by S or P, H9 is occupied by A or S, H10 isoccupied by E or V, H11 is occupied V or L, H12 is occupied by K or V,H13 is occupied by K or R, H20 is occupied by V or L, H31 is occupied byG or S, H37 is occupied by V or A, H 38 is occupied by R or K, H40 isoccupied by A or R, H48 is occupied by M or I, H60 is occupied by A orN, H64 is occupied by Q or K, H66 is occupied by R or K, H67 is occupiedby V or A, H69 is occupied by M or L, H71 is occupied by R or V, H73 isoccupied by T or I, H75 is occupied by I, S, or A, H80 is occupied by Mor V, H82a is occupied by S or T, H82b is occupied by R or S, H83 isoccupied by R or T, and H85 is occupied by D or E.
 42. The humanizedantibody of claim 41, provided positions H1, H5, H7, H9, H10, H11, H12,H13, H20, H37, H38, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a,H82b, H83, and H85 in the VH region are occupied by E, Q, P, S, V, L, V,R, L, A, K, R, I, K, A, L, V, I, S, V, T, S, T, and E, respectively. 43.The humanized antibody of claim 41, provided positions H1, H5, H11, H12,H20, H48, H69, H71, H75, H82a, H82b, H83, and H85 in the VH region areoccupied by E, Q, L, V, L, I, L, V, A, T, S, T, and E, respectively. 44.The humanized antibody of claim 41, provided positions H1, H12, H40,H48, H66, H67, H69, H71, H73, and H83 in the VH region are occupied byE, V, R, I, K, A, L, V, I, and T, respectively.
 45. The humanizedantibody of claim 41, provided positions H1, H12, H31, H40, H48, H60,H66, H67, H69, H71, H73, and H83 in the VH region are occupied by E, V,G, R, I, A, K, A, L, V, I, and T, respectively.
 46. The humanizedantibody of claim 41, provided positions H1, H12, H40, H48, H69, H73,and H83 in the VH region are occupied by E, V, R, I, L, I, and T,respectively.
 47. The humanized antibody of claim 41, provided positionsH1, H12, H31, H40, H48, H60, H66, H67, H69, H71, H73, and H83 in the VHregion are occupied by Q, V, G, R, I, A, K, A, L, V, I, and T,respectively.
 48. The humanized antibody of claim 41, provided positionsH1, H7 and H73 in the VH region are occupied by Q, P, V and I,respectively.
 49. The humanized antibody of claim 41, provided positionsH1, H12, H31, H40, H48, H60, H64, H69, H73, and H83 in the VH region areoccupied by, E, V, G, R, I, A, Q, L, I, and T, respectively.
 50. Thehumanized antibody of claim 41, provided positions H1, H5, H7, H9, H10,H11, H12, H13, H20, H48, H69, H71, H75, H82a, H82b, H83, and H85 in theVH region are occupied by E, Q, P, S, V, L, V, R, L, I, L, V, A, T, S,T, and E.
 51. The humanized antibody of claim 41, provided positions H1,H5, H11, H12, H20, H48, H66, H67, H69, H71, H73, H75, H82a, H82b, H83,and H85 in the VH region are occupied by, E, Q, L, V, L, I, K, A, L, V,I, S, T, S, T, and E.
 52. The humanized antibody of claim 41, providedpositions H1, H5, H7, H9, H10, H11, H12, H13, H20, H38, H40, H48, H66,H67, H69, H71, H73I, H75, H80, H82a, H82b, H83, and H85 in the VH regionare occupied by E, Q, P, S, V, L, V, R, L, K, R, I, K, A, L, V, I, S, V,T, S, T, and E, respectively.
 53. The humanized antibody of claim 41,provided positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H40, H48,H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83, and H85 in the VHregion are occupied by Q, P, S, V, L, V, R, L, R, I, K, A, L, V, I, S,V, T, S, T, and E, respectively.
 54. The humanized antibody of claim 41,provided positions H7, H71, and H73 in the VH region are occupied by P,V, and I, respectively.
 55. The humanized antibody of claim 41, providedposition H71 in the VH region is occupied by V.
 56. The humanizedantibody of claim 41, provided positions H1, H7, H71, and H73 in the VHregion are occupied by E, P, V, and I.
 57. (canceled)
 58. The humanizedantibody of claim 59, provided positions L4, L9, L15, L22, and L43 inthe VL region are occupied by L, S, V, S, and S, respectively.
 59. Thehumanized antibody of claim 22, wherein at least one of the followingpositions is occupied by the amino acid as specified: L4 is occupied byL, L9 is occupied by S, L15 is occupied by V, L18 is occupied by K, L19is occupied by V, L21 is occupied by M, L22 is occupied by S, and L43 isoccupied by S.
 60. The humanized antibody of claim 59, providedpositions L4, L9, L15, L18, L19, L21, L22, and L43 in the VL region areoccupied by L, S, V, K, V, M, S, and S, respectively. 61-65. (canceled)66. The humanized antibody of claim 22, comprising a mature heavy chainvariable region having an amino acid sequence at least 98% identical toany one of SEQ ID NO: 15-27 and a mature light chain variable regionhaving an amino acid sequence at least 98% identical to any one of SEQID NO: 28-30.
 67. The humanized antibody of claim 66 wherein the matureheavy chain variable region has an amino acid sequence of any of SEQ IDNO:15-27 and the mature light chain variable region has an amino acidsequence of any one of SEQ ID NO:28-30. 68-83. (canceled)
 84. Thehumanized antibody of claim 67, wherein the mature heavy chain variableregion has an amino acid sequence of SEQ ID NO:20 and the mature lightchain variable region has an amino acid sequence of SEQ ID NO:29.85-104. (canceled)
 105. The humanized antibody of claim 67, wherein themature heavy chain variable region has an amino acid sequence of SEQ IDNO:27 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:29. 106-108. (canceled)
 109. The antibody of claim3 that is an intact antibody.
 110. The antibody of claim 3 that is abinding fragment.
 111. The antibody of claim 110, wherein the bindingfragment is a single-chain antibody, Fab, or Fab′2 fragment. 112.(canceled)
 113. The antibody of claim 3, wherein the isotype is humanIgG1.
 114. The humanized antibody of claim 16, wherein the mature lightchain variable region is fused to a light chain constant region and themature heavy chain variable region is fused to a heavy chain constantregion. 115-116. (canceled)
 117. The antibody of claim 114 having atleast one mutation in the constant region.
 118. The antibody of claim117, wherein the mutation reduces complement fixation or activation bythe constant region or binding to an Fcγ receptor relative to thenatural human heavy chain constant region.
 119. The antibody of claim118 having a mutation at one or more of positions 241, 264, 265, 270,296, 297, 318, 320, 322, 329 and 331 by EU numbering or having analanine at positions 318, 320 and
 322. 120. (canceled)
 121. The antibodyof claim 3, wherein the isotype is of human IgG2 or IgG4 isotype. 122.(canceled)
 123. The antibody of claim 3, wherein the antibody isconjugated to a therapeutic, cytotoxic, cytostatic, neurotrophic, orneuroprotective agent.
 124. A pharmaceutical composition comprising anantibody as defined in claim 3 and a pharmaceutically-acceptablecarrier.
 125. A nucleic acid encoding the heavy chain and/or light chainof an antibody as described in claim 3, or a recombinant expressionvector comprising the nucleic acid or a host cell transformed with theexpression vector. 126-127. (canceled)
 128. A method of humanizing amouse antibody, the method comprising: (a) selecting one or moreacceptor antibodies; (b) identifying the amino acid residues of themouse antibody to be retained; (c) synthesizing a nucleic acid encodinga humanized heavy chain comprising CDRs of the mouse antibody heavychain and a nucleic acid encoding a humanized light chain comprisingCDRs of the mouse antibody light chain; and (d) expressing the nucleicacids in a host cell to produce a humanized antibody; wherein the mouseantibody is 16G7, wherein 16G7 is characterized by a mature heavy chainvariable region of SEQ ID NO: 7 and a mature light chain variable regionof SEQ ID NO:11.
 129. A method of producing a humanized, chimeric, orveneered antibody, the method comprising: (a) culturing cellstransformed with nucleic acids encoding the heavy and light chains ofthe antibody, so that the cells secrete the antibody; and (b) purifyingthe antibody from cell culture media; wherein the antibody is ahumanized, chimeric, or veneered form of 16G7.
 130. A method ofproducing a cell line producing a humanized, chimeric, or veneeredantibody, the method comprising: (a) introducing a vector encoding heavyand light chains of an antibody and a selectable marker into cells; (b)propagating the cells under conditions to select for cells havingincreased copy number of the vector; (c) isolating single cells from theselected cells; and (d) banking cells cloned from a single cell selectedbased on yield of antibody; wherein the antibody is a humanized,chimeric, or veneered form of 16G7.
 131. (canceled)
 132. A method ofinhibiting or reducing aggregation of tau in a subject having or at riskof developing a tau-mediated amyloidosis, comprising administering tothe subject an effective regime of the antibody of claim 3, therebyinhibiting or reducing aggregation of tau in the subject. 133.(canceled)
 134. A method of treating or effecting prophylaxis of atau-related disease in a subject, comprising administering an effectiveregime of an antibody as defined by claim 3 and thereby treating oreffecting prophylaxis of the disease. 135-137. (canceled)
 138. A methodof reducing aberrant transmission of tau comprising administering aneffective regime of an antibody as defined in claim 3 and therebyreducing transmission of tau.
 139. A method of inducing phagocytosis oftau comprising administering an effective regime of an antibody asdefined in claim 3 and thereby inducing phagocytosis of tau.
 140. Amethod of inhibiting tau aggregation or deposition comprisingadministering an effective regime of an antibody as defined in claim 3thereby inhibiting tau aggregation or deposition.
 141. A method ofinhibiting formation of tau tangles comprising administering aneffective regime of an antibody as defined in claim
 3. 142. A method ofdetecting tau protein deposits in a subject having or at risk of adisease associated with tau aggregation or deposition, comprisingadministering to a subject an antibody defined by claim 3, and detectingthe antibody bound to tau in the subject. 143-149. (canceled)
 150. Amethod of measuring efficacy of treatment in a subject being treated fora disease associated with tau aggregation or deposition, comprising (a)measuring a first level of tau protein deposits in the subject prior totreatment by administering to a subject an antibody defined by claim 3,and detecting a first amount of the antibody bound to tau in thesubject, (b) administering the treatment to the subject, (c) measuring asecond level of tau protein deposits in the subject after treatment byadministering to a subject the antibody, and detecting the antibodybound to tau in the subject, wherein a decrease in the level of tauprotein deposits indicates a positive response to treatment.
 151. Amethod of measuring efficacy of treatment in a subject being treated fora disease associated with tau aggregation or deposition, comprising (a)measuring a first level of tau protein deposits in the subject prior totreatment by administering to a subject an antibody defined by claim 3,and detecting a first amount of antibody bound to tau in the subject,(b) administering the treatment to the subject, (c) measuring a secondlevel of tau protein deposits in the subject after treatment byadministering to a subject the antibody, and detecting a second amountof antibody bound to tau in the subject, wherein no change in the levelof tau protein deposits or a small increase in tau protein depositsindicates a positive response to treatment.
 151. A method of making amonoclonal antibody that binds to the same epitope as 16G7, comprising:(a) immunizing a mouse with human tau or a fragment comprising residues113-136, (b) epitope mapping the resultant antibodies, and (c) selectingan antibody that binds to a first peptide consisting of amino acids113-127 of SEQ ID NO:1 and to a second peptide consisting of amino acids122-136 of SEQ ID NO:1, wherein binding to the second peptide is lessthan binding to the first peptide.